Compositions and methods for the modulation of jnk proteins

ABSTRACT

The invention provides compositions and methods for the treatment and diagnosis of diseases or disorders amenable to treatment through modulation of expression of a gene encoding a Jun N-terminal kinase 1 (JNK1 protein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.11/834,672 filed Aug. 6, 2007, allowed Sep. 9, 2011, which claims thebenefit of priority under 35 U.S.C. 119(e) to U.S. ProvisionalApplication No. 60/835,822, filed Aug. 4, 2006, each of which isincorporated herein by reference in its entirety.

SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledBIOL0089USC1SEQ.txt, created on Dec. 7, 2011 which is 64 Kb in size. Theinformation in the electronic format of the sequence listing isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention provides compositions and methods for detectingand modulating levels of Jun N-terminal kinases (JNK proteins), enzymeswhich are encoded by JNK genes.

BACKGROUND OF THE INVENTION

The rapid increase in the prevalence of obesity, type-2 diabetes andassociated complications is a major global health problem. Abouttwo-thirds of adults in the United States are overweight, and almostone-third are obese, according to data from the National Health andNutrition Examination Survey (NHANES) 2001 to 2004. While overweight andobesity are found worldwide, the prevalence of these conditions in theUnited States ranks high among developed nations. Overweight refers toan excess of body weight compared to set standards. The excess weightcan come from muscle, bone, fat, and/or body water. Obesity refersspecifically to having an abnormally high proportion of body fat.Individuals who are obese have a 10- to 50-percent increased risk ofdeath from all causes, compared with healthy weight individuals. Most ofthe increased risk is due to cardiovascular causes. Obesity isassociated with about 112,000 excess deaths per year in the U.S.population relative to healthy weight individuals. Obesity is a knownrisk factor for diabetes, coronary heart disease, high bloodcholesterol, stroke, hypertension, gallbladder disease, osteoarthritis,sleep apnea and other breathing problems as well as some forms of cancer(breast, colorectal, endometrial, and kidney).

Diabetes is a disorder characterized by hyperglycemia due to deficientinsulin action that can result from reduced insulin production orinsulin resistance or both. Additionally, glucotoxicity, which resultsfrom long-term hyperglycemia, induces tissue-dependent insulinresistance exacerbating the disease. Chronic hyperglycemia is also amajor risk factor for diabetes-associated complications, including heartdisease, retinopathy, nephropathy and neuropathy. Diabetes and obesity,sometimes now collectively referred to as “diabesity”) are interrelatedin that obesity is known to exacerbate the pathology of diabetes andgreater than 60% of diabetics are obese. Most human obesity isassociated with insulin resistance and leptin resistance. Obesity canhave an even greater impact on insulin action than diabetes itself.

Effective treatments are needed for diabetes, obesity, metabolicsyndrome and other diseases and conditions associated with glucoseand/or lipid metabolism and/or the disregulation thereof. The presentinvention satisfies this need and provides related advantages as well.

SUMMARY OF THE INVENTION

The present invention provides methods of reducing glucose levels in asubject by administering a therapeutically effective amount of anantisense compound targeted to a JNK1 nucleic acid. In a distinctembodiment, the invention also provides methods of reducing lipid levelsin a subject by administering a therapeutically effective amount of anantisense compound targeted to a JNK1 nucleic acid. In a furtherembodiment, methods of treating metabolic syndrome in a subject byadministering a therapeutically effective amount of an antisensecompound targeted to a JNK1 nucleic acid also are provided by thepresent invention.

In additional distinct embodiments, the invention also provides methodsof treating obesity, diabetes and metabolic syndrome in a subject. Themethods of the invention encompass administration of a therapeuticallyeffective amount of an antisense compound targeted to a JNK1 nucleicacid to a subject in need thereof.

The invention also provides methods of treating diabetes in a subject byadministering a glucose-lowering agent and a therapeutically effectiveamount of an antisense compound targeted to a JNK1 nucleic acid. Inparticular embodiments, the methods of treating diabetes in a subjectinclude administering a pharmaceutical composition encompassing aglucose-lowering agent and a therapeutically effective amount of anantisense compound targeted to a JNK1 nucleic acid.

Also provided are methods of treating diabetes, diabetes and/ormetabolic syndrome in a subject by administering a lipid lowering agentand a therapeutically effective amount of an antisense compound targetedto a JNK1 nucleic acid. In certain embodiments, the methods of treatingdiabetes in a subject include administering a pharmaceutical compositionencompassing a lipid lowering agent and a therapeutically effectiveamount of an antisense compound targeted to a JNK1 nucleic acid.

Antisense compounds useful for practicing the claimed methods, includingantisense oligonucleotides, that are complementary to SEQ ID NOS: 87,89, 90 and 91 also are provided.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which the invention(s) belong. Unless specific definitions areprovided, the nomenclature utilized in connection with, and theprocedures and techniques of, analytical chemistry, synthetic organicchemistry, and medicinal and pharmaceutical chemistry described hereinare those well known and commonly used in the art. Standard techniquescan be used for chemical synthesis, chemical analysis, pharmaceuticalpreparation, formulation and delivery, and treatment of subjects.Certain such techniques and procedures can be found for example in“Carbohydrate Modifications in Antisense Research” Edited by Sangvi andCook, American Chemical Society, Washington D.C., 1994; and “Remington'sPharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., 18thedition, 1990; and which is hereby incorporated by reference for anypurpose. Where permitted, all patents, patent applications, publishedapplications and publications, GENBANK sequences, websites and otherpublished materials referred to throughout the entire disclosure herein,unless noted otherwise, are incorporated by reference in their entirety.In the event that there is a plurality of definitions for terms herein,those in this section prevail. Where reference is made to a URL or othersuch identifier or address, it is understood that such identifiers canchange and particular information on the internet can command go, butequivalent information can be found by searching the internet. Referencethereto evidences the availability and public dissemination of suchinformation.

“Obesity” is defined as an excessively high amount of body fat oradipose tissue in relation to lean body mass. The amount of body fat (oradiposity) includes concern for both the distribution of fat throughoutthe body and the size of the adipose tissue deposits. Body fatdistribution can be estimated by skin-fold measures, waist-to-hipcircumference ratios, or techniques such as ultrasound, computedtomography, or magnetic resonance imaging. According to the Center forDisease Control and Prevention, individuals with a body mass index (BMI)of 30 or more are considered obese.

Insulin resistance is a condition in which normal amounts of insulin areinadequate to produce a normal insulin response from fat, muscle andliver cells. Insulin resistance in fat cells results in hydrolysis ofstored triglycerides, which elevates free fatty acids in the bloodplasma. Insulin resistance in muscle reduces glucose uptake whereasinsulin resistance in liver reduces glucose storage, with both effectsserving to elevate blood glucose. High plasma levels of insulin andglucose due to insulin resistance often leads to metabolic syndrome andtype 2 diabetes.

“Type 2 diabetes,” (also known as diabetes mellitus type 2, and formerlycalled diabetes mellitus type II, non-insulin-dependent diabetes(NIDDM), obesity related diabetes, or adult-onset diabetes) is ametabolic disorder that is primarily characterized by insulinresistance, relative insulin deficiency, and hyperglycemia.

A glucose tolerance test is the administration of glucose to determinehow quickly it is cleared from the blood. The test is usually used totest for diabetes, insulin resistance, and sometimes reactivehypoglycemia. The glucose is most often given orally so the common testis technically an oral glucose tolerance test (OGTT).

“Metabolic rate” refers to the amount of energy expended. Basalmetabolic rate (also known as BMR) is the amount of energy expendedwhile at rest in a neutrally temperate environment, in thepost-absorptive state (meaning that the digestive system is inactive,which requires about twelve hours of fasting in humans). The release ofenergy in this state is sufficient only for the functioning of the vitalorgans, such as the heart, lungs, brain and the rest of the nervoussystem, liver, kidneys, sex organs, muscles and skin. BMR decreases withage and with the loss of lean body mass. Increased cardiovascularexercise and muscle mass can increase BMR. Illness, previously consumedfood and beverages, environmental temperature, and stress levels canaffect one's overall energy expenditure, and can affect one's BMR asrevealed by gas analysis. It is measured when the person is at completerest, but awake. An accurate BMR measurement requires that the person'ssympathetic nervous system is not stimulated. Basal metabolic rate ismeasured under very restrictive circumstances. A more common and closelyrelated measurement, used under less strict conditions, is restingmetabolic rate (RMR). “Metabolic” and “metabolism” are terms well knowin the art and generally include the whole range of biochemicalprocesses that occur within a living organism. Metabolic disordersinclude, but are not limited to, hyperglycemia, prediabetes, diabetes(type I and type II), obesity, insulin resistance and metabolicsyndrome.

As used herein, the terms “treatment” and “treating” refer toadministering a composition of the invention to effect an alteration orimprovement of the disease or condition. Prevention, amelioration,and/or treatment can require administration of multiple doses at regularintervals, or prior to onset of the disease or condition to alter thecourse of the disease or condition. Moreover, a single agent can be usedin a single individual for each prevention, amelioration, and treatmentof a condition or disease sequentially, or concurrently.

As used herein, the term “pharmaceutical agent,” including, for example,an antisense oligonucleotide, a lipid lowering agent or a glucoselowering agent, refers to a substance provides a therapeutic benefitwhen administered to a subject. In certain embodiments, an antisenseoligonucleotide targeted to JNK1 is a pharmaceutical agent.

“Therapeutically effective amount” means an amount of a pharmaceuticalagent that provides a therapeutic benefit to a subject. In certainembodiments, a therapeutically effective amount of antisense compoundtargeted to a JNK1 nucleic acid is an amount that decreases LDL-C in thesubject.

A “pharmaceutical composition” means a mixture of substances suitablefor administering to a subject. A pharmaceutical composition cancomprise, for example, a combination of antisense oligonucleotides, acombination of antisense oligonucleotides and non-antisensepharmaceutical agents as well as the presence of a sterile aqueoussolution or other standard pharmaceitcal additive known in the art.

“Administering” means providing a pharmaceutical agent or composition toa subject, and includes, but is not limited to administering by amedical professional and self-administering. Co-administration is theadministration of two or more pharmaceutical agents to an animal. Thetwo or more pharmaceutical agents can be in a single pharmaceuticalcomposition, or can be in separate pharmaceutical compositions. Each ofthe two or more pharmaceutical agents can be administered through thesame or different routes of administration. Co-administrationencompasses administration in parallel, concomitant or sequentially.

As used herein, the term “subject” refers to an animal, including, butnot limited to a human, to whom a pharmaceutical composition isadministered. Animals include humans or non-human animal, including, butnot limited to, mice, rats, rabbits, dogs, cats, pigs, and non-humanprimates, including, but not limited to, monkeys and chimpanzees.

“Parenteral administration,” means administration through injection orinfusion. Parenteral administration includes, but is not limited to,subcutaneous administration, intravenous administration, orintramuscular administration.

Duration refers to the period of time during which an activity or eventcontinues. In certain embodiments, the duration of treatment is theperiod of time during which doses of a pharmaceutical agent areadministered.

“Subcutaneous administration” means administration just below the skin.“Intravenous administration” means administration into a vein.

Dose means a specified quantity of a pharmaceutical agent provided in asingle administration. In certain embodiments, a dose can beadministered in two or more boluses, tablets, or injections. Forexample, in certain embodiments, where subcutaneous administration isdesired, the desired dose requires a volume not easily accommodated by asingle injection. In such embodiments, two or more injections can beused to achieve the desired dose. In certain embodiments, a dose can beadministered in two or more injections to minimize injection sitereaction in a subject. Dosage unit is the form in which a pharmaceuticalagent is provided. In certain embodiments, a dosage unit is a vialcontaining lyophilized antisense oligonucleotide. In certainembodiments, a dosage unit is a vial containing reconstituted antisenseoligonucleotide.

“Metabolic syndrome” means a condition characterized by a clustering oflipid and non-lipid cardiovascular risk factors of metabolic origin. Incertain embodiments, metabolic syndrome is identified by the presence ofany 3 of the following factors: waist circumference of greater than 102cm in men or greater than 88 cm in women; serum triglyceride of at least150 mg/dL; HDL-C less than 40 mg/dL in men or less than 50 mg/dL inwomen; blood pressure of at least 130/85 mmHg; and fasting glucose of atleast 110 mg/dL. These determinants can be readily measured in clinicalpractice (JAMA, 2001, 285: 2486-2497).

As used herein, the term major risk factors refers to factors thatcontribute to a high risk for a particular disease or condition. Incertain embodiments, major risk factors for coronary heart diseaseinclude, without limitation, cigarette smoking, hypertension, low HDL-C,family history of coronary heart disease, and age.

“CHD risk factors” mean CHD risk equivalents and major risk factors.

“Coronary heart disease (CHD)” means a narrowing of the small bloodvessels that supply blood and oxygen to the heart, which is often aresult of atherosclerosis.

“Reduced coronary heart disease risk” means a reduction in thelikelihood that a subject will develop coronary heart disease. Incertain embodiments, a reduction in coronary heart disease risk ismeasured by an improvement in one or more CHD risk factors, for example,a decrease in LDL-C levels.

“Atherosclerosis” means a hardening of the arteries affecting large andmedium-sized arteries and is characterized by the presence of fattydeposits. The fatty deposits are called “atheromas” or “plaques,” whichconsist mainly of cholesterol and other fats, calcium and scar tissue,and damage the lining of arteries.

“History of coronary heart disease” means the occurrence of clinicallyevident coronary heart disease in the medical history of a subject or asubject's family member.

“Early onset coronary heart disease” means a diagnosis of coronary heartdisease prior to age 50.

“Statin intolerant individual” means a individual who as a result ofstatin therapy experiences one or more of creatine kinase increases,liver function test abnormalities, muscle aches, or central nervoussystem side effects.

“Efficacy” means the ability to produce a desired effect. For example,efficacy of a lipid-lowering therapy can be reduction in theconcentration of one or more of LDL-C, VLDL-C, IDL-C, non-HDL-C, ApoB,lipoprotein(a), or triglycerides.

“Acceptable safety profile” means a pattern of side effects that iswithin clinically acceptable limits.

“Side effects” means physiological responses attributable to a treatmentother than desired effects. In certain embodiments, side effectsinclude, without limitation, injection site reactions, liver functiontest abnormalities, renal function abnormalities, liver toxicity, renaltoxicity, central nervous system abnormalities, and myopathies. Forexample, increased aminotransferase levels in serum can indicate livertoxicity or liver function abnormality. For example, increased bilirubincan indicate liver toxicity or liver function abnormality.

“Injection site reaction” means inflammation or abnormal redness of skinat a site of injection in a subject.

“Individual compliance” means adherence to a recommended or prescribedtherapy by a subject.

“Lipid-lowering therapy” means a therapeutic regimen provided to asubject to reduce one or more lipids in a subject. In certainembodiments, a lipid-lowering therapy is provide to reduce one or moreof ApoB, total cholesterol, LDL-C, VLDL-C, IDL-C, non-HDL-C,triglycerides, small dense LDL particles, and Lp(a) in a subject.

“Lipid-lowering agent” means a pharmaceutical agent provided to asubject to achieve a lowering of lipids in the individual. For example,in certain embodiments, a lipid-lowering agent is provided to a subjectto reduce one or more of ApoB, LDL-C, total cholesterol, andtriglycerides.

“LDL-C target” means an LDL-C level that is desired followinglipid-lowering therapy.

“Comply” means the adherence with a recommended therapy by a subject.

“Recommended therapy” means a therapeutic regimen recommended by amedical professional for the treatment, amelioration, or prevention of adisease.

“Low LDL-receptor activity” means LDL-receptor activity that is notsufficiently high to maintain clinically acceptable levels of LDL-C inthe bloodstream.

“Cardiovascular outcome” means the occurrence of major adversecardiovascular events.

“Improved cardiovascular outcome” means a reduction in the occurrence ofmajor adverse cardiovascular events, or the risk thereof. Examples ofmajor adverse cardiovascular events include, without limitation, death,reinfarction, stroke, cardiogenic shock, pulmonary edema, cardiacarrest, and atrial dysrhythmia.

“Surrogate markers of cardiovascular outcome” means indirect indicatorsof cardiovascular events, or the risk thereof. For example, surrogatemarkers of cardiovascular outcome include carotid intimal mediathickness (CIMT). Another example of a surrogate marker ofcardiovascular outcome includes atheroma size. Atheroma size can bedetermined by intravascular ultrasound (IVUS).

“Increased HDL-C” means an increase in serum HDL-C in a subject overtime.

“Lipid-lowering” means a reduction in one or more serum lipids in asubject over time.

“Therapeutic lifestyle change” means dietary and lifestyle changesintended to lower cholesterol and reduce the risk of developing heartdisease, and includes recommendations for dietary intake of total dailycalories, total fat, saturated fat, polyunsaturated fat, monounsaturatedfat, carbohydrate, protein, cholesterol, insoluble fiber, as well asrecommendations for physical activity.

“Statin” means a pharmaceutical agent that inhibits the activity ofHMG-CoA reductase.

“HMG-CoA reductase inhibitor” means a pharmaceutical agent that actsthrough the inhibition of the enzyme HMG-CoA reductase.

“Cholesterol absorption inhibitor” means a pharmaceutical agent thatinhibits the absorption of exogenous cholesterol obtained from diet.

“LDL apheresis” means a form of apheresis by which LDL-C is removed fromblood. Typically, a subject's blood is removed from a vein, andseparated into red cells and plasma. LDL-C is filtered out of the plasmaprior to return of the plasma and red blood cells to the individual.

“MTP inhibitor” means a pharmaceutical agent that inhibits the enzymemicrosomal triglyceride transfer protein.

“Low density lipoprotein-cholesterol (LDL-C)” means cholesterol carriedin low density lipoprotein particles. Concentration of LDL-C in serum(or plasma) is typically quantified in mg/dL or nmol/L. “Serum LDL-C”and “plasma LDL-C” mean LDL-C in the serum and plasma, respectively.

“Very low density lipoprotein-cholesterol (VLDL-C)” means cholesterolassociated with very low density lipoprotein particles. Concentration ofVLDL-C in serum (or plasma) is typically quantified in mg/dL or nmol/L.“Serum VLDL-C” and “plasma VLDL-C” mean VLDL-C in the serum or plasma,respectively.

“Intermediate low density lipoprotein-cholesterol (IDL-C)” meanscholesterol associated with intermediate density lipoprotein.Concentration of IDL-C in serum (or plasma) is typically quantified inmg/mL or nmol/L. “Serum IDL-C” and “plasma IDL-C” mean IDL-C in theserum or plasma, respectively.

“Non-high density lipoprotein-cholesterol (Non-HDL-C)” means cholesterolassociated with lipoproteins other than high density lipoproteins, andincludes, without limitation, LDL-C, VLDL-C, and IDL-C.

“High density lipoprotein-C (HDL-C)” means cholesterol associated withhigh density lipoprotein particles. Concentration of HDL-C in serum (orplasma) is typically quantified in mg/dL or nmol/L. “Serum HDL-C” and“plasma HDL-C” mean HDL-C in the serum and plasma, respectively.

“Total cholesterol” means all types of cholesterol, including, but notlimited to, LDL-C, HDL-C, IDL-C and VLDL-C. Concentration of totalcholesterol in serum (or plasma) is typically quantified in mg/dL ornmol/L.

“Lipoprotein(a)” or “Lp(a)” means a lipoprotein particle that iscomprised of LDL-C, an apolipoprotein(a) particle, and anapolipoproteinB-100 particle.

“ApoA1” means apolipoprotein-A1 protein in serum. Concentration of ApoA1in serum is typically quantified in mg/dL or nmol/L.

“ApoB:ApoA1 ratio” means the ratio of ApoB concentration to ApoA1concentration.

“ApoB-containing lipoprotein” means any lipoprotein that hasapolipoprotein B as its protein component, and is understood to includeLDL, VLDL, IDL, and lipoprotein(a).

“Small LDL particle” means a subclass of LDL particles characterized bya smaller, denser size compared to other LDL particles. In certainembodiments, intermediate LDL particles are 23-27 nm in diameter. Incertain embodiments, large LDL particles are 21.2-23 nm in diameter. Incertain embodiments, small LDL particles are 18-21.2 nm in diameter. Incertain embodiments, particle size is measured by nuclear magneticresonance analysis.

“Small VLDL particle” means a subclass of VLDL particles characterizedby a smaller, denser size compared to other VLDL particles. In certainembodiments, large VLDL particles are greater than 60 nm in diameter. Incertain embodiments, medium VLDL particles are 35-60 nm in diameter. Incertain embodiments, small VLDL particles are 27-35 nm in diameter. Incertain embodiments, particle size is measured by nuclear magneticresonance analysis.

“Triglycerides” means lipids that are the triesters of glycerol. “Serumtriglycerides” mean triglycerides present in serum. “Livertriglycerides” mean triglycerides present in liver tissue.

“Serum lipids” mean cholesterol and triglycerides in the serum.

“Elevated total cholesterol” means total cholesterol at a concentrationin a subject at which lipid-lowering therapy is recommended, andincludes, without limitation, elevated LDL-C″, “elevated VLDL-C,”“elevated IDL-C” and “elevated non-HDL-C.” In certain embodiments, totalcholesterol concentrations of less than 200 mg/dL, 200-239 mg/dL, andgreater than 240 mg/dL are considered desirable, borderline high, andhigh, respectively. In certain embodiments, LDL-C concentrations of 100mg/dL, 100-129 mg/dL, 130-159 mg/dL, 160-189 mg/dL, and greater than 190mg/dL are considered optimal, near optimal/above optimal, borderlinehigh, high, and very high, respectively.

“Elevated triglyceride” means concentrations of triglyceride in theserum or liver at which lipid-lowering therapy is recommended, andincludes “elevated serum triglyceride” and “elevated livertriglyceride.” In certain embodiments, serum triglyceride concentrationof 150-199 mg/dL, 200-499 mg/dL, and greater than or equal to 500 mg/dLis considered borderline high, high, and very high, respectively.

“Elevated small LDL particles” means a concentration of small LDLparticles in a subject at which lipid-lowering therapy is recommended.

“Elevated small VLDL particles” means a concentration of small VLDLparticles in a subject at which lipid-lowering therapy is recommended.

“Elevated lipoprotein(a)” means a concentration of lipoprotein(a) in asubject at which lipid-lowering therapy is recommended.

“Low HDL-C” means a concentration of HDL-C in a subject at whichlipid-lowering therapy is recommended. In certain embodimentslipid-lowering therapy is recommended when low HDL-C is accompanied byelevations in non-HDL-C and/or elevations in triglyceride. In certainembodiments, HDL-C concentrations of less than 40 mg/dL are consideredlow. In certain embodiments, HDL-C concentrations of less than 50 mg/dLare considered low.

“ApoB” means apolipoprotein B-100 protein. Concentration of ApoB inserum (or plasma) is typically quantified in mg/dL or nmol/L. “SerumApoB” and “plasma ApoB” mean ApoB in the serum and plasma, respectively.

“LDL/HDL ratio” means the ratio of LDL-C to HDL-C.

“Oxidized-LDL” or “Ox-LDL-C” means LDL-C that is oxidized followingexposure to free radicals.

“Individual having elevated LDL-C levels” means a subject who has beenidentified by a medical professional (e.g. a physician) as having LDL-Clevels near or above the level at which therapeutic intervention isrecommended, according to guidelines recognized by medicalprofessionals. Such a subject can also be considered “in need oftreatment” to decrease LDL-C levels.

“Individual having elevated apoB-100 levels” means a subject who hasbeen identified as having apoB-100 levels near or below the level atwhich therapeutic intervention is recommended, according to guidelinesrecognized by medical professionals. Such a subject can also beconsidered “in need of treatment” to decrease apoB-100 levels.

“Treatment of elevated LDL-C levels” means administration of anantisense compound targeted to a JNK1 nucleic acid to a subject havingelevated LDL-C levels.

“Treatment of atherosclerosis” means administration of an antisensecompound targeted to a JNK1 nucleic acid to a subject who, based upon aphysician's assessment, has or is likely to have atherosclerosis.“Prevention of atherosclerosis” means administration of an antisensecompound targeted to a JNK1 nucleic acid to a subject who, based upon aphysician's assessment, is susceptible to atherosclerosis.

As used herein, the term “modulation” refers to a perturbation offunction or activity when compared to the level of the function oractivity prior to modulation. For example, modulation includes thechange, either an increase (stimulation or induction) or a decrease(inhibition or reduction) in gene expression. As further example,modulation of expression can include perturbing splice site selection ofpre-mRNA processing.

As used herein, the term “expression” refers to all the functions andsteps by which a gene's coded information is converted into structurespresent and operating in a cell. Such structures include, but are notlimited to the products of transcription and translation.

“Antisense inhibition” means reduction of a target nucleic acid levelsin the presence of an antisense compound complementary to a targetnucleic acid compared to target nucleic acid levels in the absence ofthe antisense compound.

As used herein, the term “target” refers to a protein, the modulation ofwhich is desired.

As used herein, the term “target gene” refers to a gene encoding atarget.

“Targeting” means the process of design and selection of an antisensecompound that will specifically hybridize to a target nucleic acid or aparticular region of nucleotides within a target nucleic acid moleculeand induce a desired effect.

“Targeted” means having a nucleobase sequence that will allow specifichybridization of an antisense compound to a target nucleic acid or aparticular region of nucleotides within a target nucleic acid moleculeto induce a desired effect. In certain embodiments, a desired effect isreduction of a target nucleic acid. In certain such embodiments, adesired effect is reduction of JNK1 mRNA.

As used herein, the terms “target nucleic acid,” “target RNA,” “targetRNA transcript,” “nucleic acid target” and “nucleic acid moleculeencoding a target” refer to any nucleic acid molecule the expression oractivity of which is capable of being modulated by an antisensecompound. Target nucleic acids include, but are not limited to, RNA(including, but not limited to pre-mRNA and mRNA or portions thereof)transcribed from DNA encoding a target, and also cDNA derived from suchRNA, and miRNA. For example, the target nucleic acid can be a cellulargene (or mRNA transcribed from the gene) whose expression is associatedwith a particular disorder or disease state, or a nucleic acid moleculefrom an infectious agent.

A “JNK1 nucleic acid” means any nucleic acid encoding JNK1. For example,in certain embodiments, a JNK1 nucleic acid includes, withoutlimitation, a DNA sequence encoding JNK1, an RNA sequence transcribedfrom DNA encoding JNK1, and an mRNA sequence encoding JNK1. “JNK1 mRNA”means an mRNA encoding a JNK1 protein.

As used herein, the term “5′ target site” refers to the nucleotide of atarget nucleic acid which is complementary to the 5′-most nucleotide ofa particular antisense compound.

As used herein, the term “3′ target site” refers to the nucleotide of atarget nucleic acid which is complementary to the 3′-most nucleotide ofa particular antisense compound.

As used herein, the term “target region,” refers to a portion of atarget nucleic acid to which one or more antisense compounds iscomplementary.

As used herein, the term “target segment” refers to a smaller orsub-portions of a region within a target nucleic acid.

As used herein, the term “complementarity” refers to the ability of anucleobase to base pair with another nucleobase. For example, in DNA,adenine (A) is complementary to thymine (T). For example, in RNA,adenine (A) is complementary to uracil (U). In certain embodiments,complementary nucleobase refers to a nucleobase of an antisense compoundthat is capable of base pairing with a nucleobase of its target nucleicacid. For example, if a nucleobase at a certain position of an antisensecompound is capable of hydrogen bonding with a nucleobase at a certainposition of a target nucleic acid, then the position of hydrogen bondingbetween the oligonucleotide and the target nucleic acid is considered tobe complementary at that nucleobase pair.

As used herein, the term “non-complementary nucleobase” refers to anucleobase that does not form hydrogen bonds with another nucleobase orotherwise support hybridization.

As used herein, the term “complementary” refers to the capacity of anoligomeric compound to hybridize to another oligomeric compound ornucleic acid through base pairing. In certain embodiments, an antisensecompound and its target are complementary to each other when asufficient number of corresponding positions in each molecule areoccupied by nucleobases that can pair with each other to allow stableassociation between the antisense compound and the target. One skilledin the art recognizes that the inclusion of mismatches is possiblewithout eliminating the ability of the oligomeric compounds to remain inassociation. Therefore, described herein are antisense compounds thatcan comprise up to about 20% nucleotides that are mismatched (i.e., arenot nucleobase complementary to the corresponding nucleotides of thetarget). Preferably the antisense compounds contain no more than about15%, more preferably not more than about 10%, most preferably not morethan 5% or no mismatches. The remaining nucleotides are nucleobasecomplementary or otherwise do not disrupt hybridization (e.g., universalbases). One of ordinary skill in the art would recognize the compoundsprovided herein are at least 80%, at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, at least 99% or 100%complementary to a target nucleic acid.

As used herein, the term “mismatch” refers to a non-complementarynucleobase within a complementary oligomeric compound.

As used herein, “hybridization” means the pairing of complementaryoligomeric compounds (e.g., an antisense compound and its target nucleicacid). While not limited to a particular mechanism, the most commonmechanism of pairing involves hydrogen bonding, which can beWatson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, betweencomplementary nucleoside or nucleotide bases (nucleobases). For example,the natural base adenine is nucleobase complementary to the naturalnucleobases thymidine and uracil which pair through the formation ofhydrogen bonds. The natural base guanine is nucleobase complementary tothe natural bases cytosine and 5-methyl cytosine. Hybridization canoccur under varying circumstances.

As used herein, the term “specifically hybridizes” refers to the abilityof an oligomeric compound to hybridize to one nucleic acid site withgreater affinity than it hybridizes to another nucleic acid site. Incertain embodiments, an antisense oligonucleotide specificallyhybridizes to more than one target site.

As used herein, “designing” or “designed to” refer to the process ofdesigning an oligomeric compound that specifically hybridizes with aselected nucleic acid molecule.

“Portion” means a defined number of contiguous (i.e. linked) nucleobasesof a target nucleic acid. In certain embodiments, a portion is a definednumber of contiguous nucleobases of a target nucleic acid. In certainembodiments, a portion is a defined number of contiguous nucleobases ofan antisense compound.

As used herein, the term “oligomeric compound” refers to a polymericstructure comprising two or more sub-structures and capable ofhybridizing to a region of a nucleic acid molecule. In certainembodiments, oligomeric compounds are oligonucleosides. In certainembodiments, oligomeric compounds are oligonucleotides. In certainembodiments, oligomeric compounds are antisense compounds. In certainembodiments, oligomeric compounds are antisense oligonucleotides. Incertain embodiments, oligomeric compounds are chimeric oligonucleotides.

As used herein, the term “antisense compound” refers to an oligomericcompound that is at least partially complementary to a target nucleicacid molecule to which it hybridizes. In certain embodiments, anantisense compound modulates (increases or decreases) expression of atarget nucleic acid. Antisense compounds include, but are not limitedto, compounds that are oligonucleotides, oligonucleosides,oligonucleotide analogs, oligonucleotide mimetics, and chimericcombinations of these. Consequently, while all antisense compounds areoligomeric compounds, not all oligomeric compounds are antisensecompounds.

As used herein, the term “oligonucleotide” refers to an oligomericcompound comprising a plurality of linked nucleotides. In certainembodiment, one or more nucleotides of an oligonucleotide is modified.In certain embodiments, an oligonucleotide contains ribonucleic acid(RNA) or deoxyribonucleic acid (DNA). In certain embodiments,oligonucleotides are composed of naturally- and/ornon-naturally-occurring nucleobases, sugars and covalent internucleotidelinkages, and can further include non-nucleic acid conjugates.

“Oligonucleoside” means an oligonucleotide in which the internucleosidelinkages do not contain a phosphorus atom.

“Antisense oligonucleotide” means a single-stranded oligonucleotidehaving a nucleobase sequence that will permits hybridization to acorresponding region of a target nucleic acid.

“Motif” means the pattern of unmodified and modified nucleosides in anantisense compound.

“Chimeric antisense compounds” means an antisense compounds that have atleast 2 chemically distinct regions, each region having a plurality ofsubunits.

As used herein, the term “gapmer” refers to a chimeric oligomericcompound comprising a central region (a “gap”) and a region on eitherside of the central region (the “wings”), wherein the gap comprises atleast one modification that is different from that of each wing. Suchmodifications include nucleobase, monomeric linkage, and sugarmodifications as well as the absence of modification (unmodified). Thegap region generally supports RNaseH cleavage.

As used herein, the term “nucleoside” means a glycosylamine comprising anucleobase and a sugar. Nucleosides includes, but are not limited to,naturally occurring nucleosides, abasic nucleosides, modifiednucleosides, and nucleosides having mimetic bases and/or sugar groups.

As used herein, the term “nucleotide” refers to a glycosomine comprisinga nucleobase and a sugar having a phosphate group covalently linked tothe sugar. Nucleotides can be modified with any of a variety ofsubstituents.

As used herein, the term “nucleobase” refers to the base portion of anucleoside or nucleotide. A nucleobase can comprise any atom or group ofatoms capable of hydrogen bonding to a base of another nucleic acid.

As used herein, the term “monomer” refers to a single unit of anoligomer. Monomers include, but are not limited to, nucleosides andnucleotides, whether naturally occurring or modified.

As used herein, the term “deoxyribonucleotide” means a nucleotide havinga hydrogen at the 2′ position of the sugar portion of the nucleotide.Deoxyribonucleotides can be modified with any of a variety ofsubstituents.

As used herein, the term “ribonucleotide” means a nucleotide having ahydroxy at the 2′ position of the sugar portion of the nucleotide.Ribonucleotides can be modified with any of a variety of substituents.

“Unmodified nucleotide” means a nucleotide composed of naturallyoccurring nucleobases, sugar moieties and internucleoside linkages. Incertain embodiments, an unmodified nucleotide is an RNA nucleotide(i.e., β-D-ribonucleosides) or a DNA nucleotide (i.e.,β-D-deoxyribonucleoside).

“Modified nucleotide” means a nucleotide having, independently, amodified sugar moiety, modified internucleoside linkage, or modifiednucleobase. A “modified nucleoside” means a nucleotide having,independently, a modified sugar moiety or modified nucleobase.

“Internucleoside linkage” means a covalent linkage between adjacentnucleosides.

“Naturally occurring internucleoside linkage” means a 3′ to 5′phosphodiester linkage.

“Modified internucleoside linkage” means substitution and/or any changefrom a naturally occurring internucleoside linkage

“Modified sugar moiety” means substitution and/or any change from anatural sugar moiety. For the purposes of this disclosure, a “naturalsugar moiety” is a sugar moiety found in DNA (2′-H) or RNA (2′-OH).

“Modified nucleobase” means any nucleobase other than adenine, cytosine,guanine, thymidine, or uracil. An “unmodified nucleobase” means thepurine bases adenine (A) and guanine (G), and the pyrimidine basesthymine (T), cytosine (C) and uracil (U).

“Natural sugar moiety” means a sugar moiety found in DNA (2′-H) or RNA(2′-OH).

“2′-O-methoxyethyl sugar moiety” means a 2′-substituted furanosyl ringhaving a 2′-O(CH2)2-OCH3 (2′-O-methoxyethyl or 2′-MOE) substituentgroup.

“2′-O-methoxyethyl nucleotide” means a nucleotide comprising a2′-O-methoxyethyl modified sugar moiety.

“Bicyclic nucleic acid sugar moiety” means a furanosyl ring modified bythe bridging of two non-geminal ring atoms.

“Prodrug” means a therapeutic agent that is prepared in an inactive formthat is converted to an active form (i.e., drug) within the body orcells thereof by the action of endogenous enzymes or other chemicalsand/or conditions.

“Pharmaceutically acceptable salts” means physiologically andpharmaceutically acceptable salts of antisense compounds, i.e., saltsthat retain the desired biological activity of the parentoligonucleotide and do not impart undesired toxicological effectsthereto.

“Cap structure” or “terminal cap moiety” means chemical modifications,which have been incorporated at either terminus of an antisensecompound.

Overview

In the context of the invention, the terms “Jun N-terminal kinase,”“c-Jun N-terminal kinase” and “JNK1 protein” refer to proteins actuallyknown to phosphorylate the amino terminal (N-terminal) portion of theJun subunit of AP-1, as well as those that have been tentativelyidentified as JNK1 proteins based on amino acid sequence but which canin fact additionally or alternatively bind and/or phosphorylate eitherother transcription factors (e.g., ATF2) or kinase substrates that arenot known to be involved in transcription (Derijard et al., Cell, 1994,76, 1025; Kallunki et al., Genes & Development, 1994, 8, 2996; Gutta etal., EMBO J., 1996, 15, 2760).

AP-1 is one member of a family of related heterodimeric transcriptionfactor complexes found in eukaryotic cells or viruses (The FOR and JUNFamilies of Transcription Factors, Angel and Hairlike, Eds., CBC Press,Boca Raton, Fla., 1994; Bohmann et al., Science, 1987, 238, 1386; Angelet al., Nature, 1988, 332, 166). Two relatively well-characterized AP-1subunits are c-For and c-Jun; these two proteins are products of thec-for and c-jun proto-oncogenes, respectively. (Rahmsdorf, Chapter 13,and Rapp et al., Chapter 16 In: The FOS and JUN Families ofTranscription Factors, Angel and Herrlich, Eds., CBC Press, Boca Raton,Fla., 1994)

The phosphorylation of proteins plays a key role in the transduction ofextracellular signals into the cell. Typical MAP kinase pathways areknown and recited in the literature. (See e.g., Cano et al., TrendsBiochem. Sci., 1995, 20, 117 Cobb et al., J. Biol. Chem., 1995, 270,14843; Seger et al., FASEB J., 1995, 9, 726; Cano et al., TrendsBiochem. Sci., 1995, 20, 117).

One of the signal transduction pathways involves the MAP kinases JunN-terminal kinase 1 (JNK1) and Jun N-terminal kinase 2 (JNK2) which areresponsible for the phosphorylation of specific sites (Serine 63 andSerine 73) on the amino terminal portion of c-Jun. Phosphorylation ofthese sites potentiates the ability of AP-1 to activate transcription(Binetruy et al., Nature, 1991, 351, 122; Smeal et al., Nature, 1991,354, 494). Besides JNK1 and JNK2, other JNK family members have beendescribed, including JNK3 (Gutta et al., EMBO J., 1996, 15, 2760),initially named p49^(3F12) kinase (Mohit et al., Neuron, 1994, 14, 67).

Recent studies have indicated that JNKs interfere with insulin action incultured cells and are activated by free fatty acids and inflammatorycytokines; both implicated in the development of type-2 diabetes. Thus,JNK can be a mediator of obesity and insulin resistance. (Hirosumi etal., Nature, 2002, 420:333-336).

JNKs or c-Jun N-terminal kinases are a family of serine/threonineprotein kinases of the mitogen-activated protein kinase (MAPK) group andare involved in a variety of physiological functions. They are activatedin response to different stimuli which cause cellular stress includingheat shock, irradiation, hypoxia, chemotoxins, peroxides, and somecytokines (Bennett et al., 2003; Bogoyevitch et al., 2004). Obesity alsocauses cellular stress due to mechanical changes, excess lipidaccumulation, abnormalities in intracellular energy fluxes, and alterednutrient availability, as well as changed plasma levels of cytokines(Hotamisligil, 2005; Ozcan et al., 2004; Waetzig and Herdegen, 2005).JNK activity is much higher in liver, fat and muscle in both geneticallyobese (ob/ob) mice and diet-induced obese (DIO) mice than in theirrespective controls (Hirosumi et al., 2002; Ozcan et al., 2004).

Obesity is considered a long-term disease. There are over thirty seriousmedical concerns related to obesity. Metabolic syndrome is a combinationof medical disorders that increase one's risk for cardiovascular diseaseand diabetes. The symptoms, including high blood pressure, hightriglycerides, decreased HDL and obesity, tend to appear together insome individuals. It affects a large number of people in a clusteredfashion. In some studies, the prevalence in the USA is calculated asbeing up to 25% of the population. Metabolic syndrome is known undervarious other names, such as (metabolic) syndrome X, insulin resistancesyndrome, Reaven's syndrome or CHAOS.

The present invention is based, in part, on the discovery of antisensecompounds that target nucleic acid encoding JNK1 and which function toreduce JNK1 levels in a subject.

Effective treatments are needed for diabetes, obesity, metabolicsyndrome and other diseases and conditions associated with glucoseand/or lipid metabolism and/or the disregulation thereof. Certaincompounds on the market for the treatment of diabetes are known toinduce weight gain, a very undesirable side effect to the treatment ofthis disease. Therefore, a compound that has the potential to treat bothdiabetes and obesity would provide a significant improvement overcurrent treatments.

A role for JNK1 in both insulin resistance and obesity is identified andJNK1 is presented herein as a therapeutic target for a range ofmetabolic diseases and conditions, including diabetes, obesity andmetabolic syndrome. Therefore, provided herein are compounds andcompositions targeting JNK1 and methods for the treatment of metabolicdiseases and conditions. Metabolic conditions are characterized by analteration or disturbance in metabolic function.

In accordance with the present invention, oligonucleotides are providedwhich specifically hybridize with a nucleic acid encoding a JNK1protein. Certain oligonucleotides of the invention are designed to bindeither directly to mRNA transcribed from, or to a selected DNA portionof, a JNK1 gene that encodes a JNK1 protein, thereby modulating theexpression thereof and/or the phosphorylation of one or more substratesfor the JNK1 protein. Pharmaceutical compositions comprising theoligonucleotides of the invention, and various methods of using theoligonucleotides of the invention, including methods of modulating oneor more metastatic events, are also herein provided.

Provided herein are methods, compounds and compositions for modulatingJNK1 expression in a subject. In certain embodiments, methods, compoundsand compositions are provided for reducing JNK1 levels, expression,and/or activity in a subject. In certain embodiments the reduction ofJNK1 expression, activity and/or nucleic acid levels occurs in liver andfat tissues of a subject. In certain embodiments the subject is ananimal. In certain embodiments the animal is a human.

Provided herein are methods, compounds and compositions for thetreatment, prevention and/or amelioration of diseases or conditionsassociated with glucose and/or lipid metabolism and/or the disregulationthereof. In certain embodiments, the methods, compounds and compositionsare for the treatment, prevention and/or amelioration of diabetes,obesity and metabolic syndrome. In certain embodiments, the methods,compounds and compositions are for the treatment, prevention and/oramelioration of hypercholesterolemia, mixed dyslipidemia,atherosclerosis, a risk of developing atherosclerosis, coronary heartdisease, a history of coronary heart disease, early onset coronary heartdisease, one or more risk factors for coronary heart disease, type IIdiabetes, type II diabetes with dyslipidemia, dyslipidemia,hypertriglyceridemia, hyperlipidemia, hyperfattyacidemia, hepaticsteatosis, non-alcoholic steatohepatitis, or non-alcoholic fatty liverdisease. In certain embodiments, such methods, compounds andcompositions are used to treat, slow, prevent, delay or ameliorate thesequelae of diabetes including, but not limited to, retinopathy,neuropathy, cardiovascular complications and nephropathy.

Provided herein are methods, compounds and compositions for improvingblood glucose control or tolerance. In certain embodiments, the methods,compounds and compositions are for improving insulin sensitivity. Alsoprovided are methods, compounds and compositions for the reduction ofglucose levels. In certain embodiments, such glucose levels can beblood, plasma and/or serum glucose levels. In certain embodiments, suchglucose levels can be fed or fasting glucose levels

Also provided are methods, compounds and compositions for the reductionof lipids. Also provided are methods, compounds and compositions for thereduction of triglyceride levels in a subject. In certain embodiments,such triglyceride levels are plasma triglyceride levels. In certainembodiments, such triglyceride levels are liver triglyceride levels.Also provided are methods of improving liver steatosis. Also providedare methods, compounds and compositions for the reduction of cholesterollevels. In certain embodiments, such cholesterol levels are plasmacholesterol levels.

Also provided are methods, compounds and compositions for modulatingexpression of metabolic and/or lipogenic genes. In certain embodiments,the metabolic and/or lipgenic genes listed in Table 35 below. In certainembodiments expression levels of one or more of ACC1, ACC2, FAS, SCD1and DGAT1, DGAT2, RBP4, G6Pase and PKCε are lowered. In certainembodiments, levels are reduced by about 30% to about 70%. Also providedare methods, compounds and compositions for lowering lipogenesis. Incertain embodiments lipogenesis is lowered by lowering expression ofsuch metabolic or lipogenic genes. In certain embodiments, expressionlevels of ARβ₃, UCP1, UCP2 and PPARα are increased. In certainembodiments, levels are increased by up to about 70%. Such methodsinclude administering to a subject an antisense compound targeted to anucleic acid encoding JNK1. In certain embodiments, such methods includethe administration of a therapeutically effective amount of an antisensecompound targeted to a JNK1 nucleic acid. In certain embodiments, thecompound is administered in a composition. In certain embodiments thesubject is an animal. In certain embodiments the animal is a human. Incertain embodiments, the subject to which the antisense compound isadministered and in which levels are modulated has one or more of thediseases or disorders listed above. In certain embodiments, the subjectto which the antisense compound is administered and in which levels arelowered has obesity, hypercholesterolemia, mixed dyslipidemia,atherosclerosis, coronary heart disease, diabetes, type II diabetes,type II diabetes with dyslipidemia, dyslipidemia, hypertriglyceridemia,hyperlipidemia, hyperfattyacidemia, hepatic steatosis, non-alcoholicsteatohepatitis (NASH), or non-alcoholic fatty liver disease (NAFL).

In certain embodiments, the subject to which the antisense compound isadministered has elevated glucose levels, triglyceride levels orcholesterol levels or any combination thereof. In certain embodiments,such glucose levels can be blood, plasma and/or serum glucose levels. Incertain embodiments, such glucose levels can be fed or fasting glucoselevels. In certain embodiments, such glucose levels are fed or fastingblood glucose levels. In certain embodiments, such triglyceride levelsare plasma triglyceride levels. In certain embodiments, suchtriglyceride levels are liver triglyceride levels. In certainembodiments, such cholesterol levels are plasma cholesterol levels. Incertain embodiments, the administration thereby reduces glucose levels,triglyceride levels or cholesterol levels. In certain embodiments thesubject is an animal. In certain embodiments the animal is a human.

Also provided are methods for reducing serum glucose levels, serumtriglyceride levels or plasma cholesterol levels in a subject whichinclude selecting a subject having elevated serum glucose levels, serumtriglyceride levels or plasma cholesterol levels, and administering tothe individual a therapeutically effective amount of an antisensecompound targeted to a JNK1 nucleic acid, and additionally monitoringserum glucose levels, serum triglyceride levels or plasma cholesterollevels. In certain embodiments the individual is an animal. In certainembodiments the individual is a human.

Further provided are methods for treating, preventing and/orameliorating diabetes, obesity or metabolic syndrome, or another diseaseor condition associated with glucose and/or lipid metabolism and/or thedisregulation thereof, in a subject. Such method includes selecting asubject diagnosed with diabetes, obesity or metabolic syndrome or otherdisease or condition associated with glucose and/or lipid metabolism,administering to the individual a therapeutically effective amount of anantisense compound targeted to a JNK1 nucleic acid, and monitoringfactors related to diabetes, obesity or metabolic syndrome or otherrelated disease or condition.

Further provided are methods of increasing metabolic rate. Also providedare methods for lowering body weight gain. Also provided are methods forlowering epididymal fat pad weight. Also provided are methods forlowering whole body fat content. Such methods include administering to asubject an antisense compound targeted to a nucleic acid encoding JNK1.In certain embodiments, such methods include the administration of atherapeutically effective amount of an antisense compound targeted to aJNK1 nucleic acid. In certain embodiments, the compound is administeredin a composition. In certain embodiments the subject is an animal. Incertain embodiments the animal is a human. In certain embodiments, thesubject to which the antisense compound is administered and in whichmetabolic rate is increased and/or weight or fat content is lowered hasone or more of the diseases or disorders listed above. In certainembodiments, the subject to which the antisense compound is administeredand in which metabolic rate is increased and/or weight or fat content islowered has obesity, diabetes or metabolic syndrome.

It is understood that the terms individual and subject are usedinterchangeably herein and that any of the methods provided herein canbe useful for a subject or a subject and that subject or individual canbe an animal and particularly a human.

Any of the methods provided herein can further comprise monitoring serumor plasma glucose levels, serum or plasma triglyceride levels or serumor plasma cholesterol levels.

In any of the aforementioned methods, administration of the antisensecompound can comprise parenteral administration. The parenteraladministration can further comprise subcutaneous or intravenousadministration.

In any of the compounds, compositions or methods provided herein, theantisense compound can have least 80%, at least 90%, or at least 95%complementarity to SEQ ID NO: 87, 89, 90 or 91. Alternatively, theantisense compound can have 100% complementarity to SEQ ID NO: 87, 89,90 or 91.

The antisense compounds provided herein and employed in any of thedescribed methods can be 8 to 80 subunits in length, 12 to 50 subunitsin length, 12 to 30 subunits in length, 15 to 30 subunits in length, 18to 24 subunits in length, 19 to 22 subunits in length, or 20 subunits inlength. Further, the antisense compounds employed in any of thedescribed methods can be antisense oligonucleotides 8 to 80 nucleotidesin length, 12 to 50 nucleotides in length, 12 to 30 nucleotides inlength 15 to 30 nucleotides in length, 18 to 24 nucleotides in length,19 to 22 nucleotides in length, or 20 nucleotides in length.

In any of the compounds, compositions and methods provided, theantisense compound can be an antisense oligonucleotide. Moreover, theantisense oligonucleotide can be chimeric. The chimeric antisenseoligonucleotide can be a gapmer antisense oligonucleotide. The gapmerantisense oligonucleotide can comprise a gap segment of ten2′-deoxynucleotides positioned between wing segments of five 2′-MOEnucleotides.

In any of the compounds, compositions and methods provided, theantisense compounds can have at least one modified internucleosidelinkage. Additionally, each internucleoside linkage can be aphosphorothioate internucleoside linkage. Each cytosine can be a5-methyl cytosine.

A compound for treatment of obesity and metabolic syndrome can be anantisense compound 12 to 30 nucleobases targeted to a JNK1 nucleic acid.The compound can have at least 70% to 100% complementarity to any of SEQID Nos: 87, 89, 90 or 91. The antisense oligonucleotide can be a gapmerantisense oligonucleotide. The gapmer antisense oligonucleotide cancomprise a gap segment of ten 2′-deoxynucleotides positioned betweenwing segments of five 2′-MOE nucleotides.

The antisense compounds can have at least one modified internucleosidelinkage. Additionally, each internucleoside linkage can be aphosphorothioate internucleoside linkage. Each cytosine can be a5-methyl cytosine.

Both genetic and dietary mouse models of obesity were treated with JNK1ASO. JNK1 ASO treatment markedly and specifically reduced the geneexpression of JNK1 in both liver and fat tissues, which resulted in adramatic reduction of JNK1 activity in these tissues. The treatmentlowered BW (or body weight gain), fat depot weight and whole body fatcontent, and increased metabolic rate without causing liver toxicity orother side-effects as compared to controls. The treatment markedlylowered fed and fasting plasma glucose and insulin levels, improvedglucose and insulin tolerance, improved liver steatosis and loweredplasma cholesterol levels. These data indicate that specific inhibitionof JNK1 expression and activity with ASO in the two major metabolictissues improved adiposity and related metabolic disorders in thesemodels.

Treatment also resulted in improved feed efficiency. Additionally, anincreased metabolic rate in the ASO-treated mice was confirmed byindirect calorimetry. Quantitative RT-PCR analysis found increased geneexpression in BAT from these mice of both ARβ₃ and UCP1, two key genesinvolved in catabolism and fuel combustion in rodents. Increasedexpression of PPARα, UCP2, and UCP3, and decreased expression of ACC2were also found in either liver or WAT, leading further support to thefinding of an increased metabolic rate. In addition, an extensive andprofound decrease in the expression of lipogenic genes and unchangedexpression of two key lipolytic genes, HSL and ATGL, were found in WAT,indicating decreased lipogenesis and unchanged lipolysis with reductionof JNK1 expression in this tissue. Marked decrease in expression of ACC1and FAS, two key genes involved in de novo fatty acid synthesis, inliver was also detected. Furthermore, increased fatty acid oxidation anddecreased de novo fatty acid synthesis were directly demonstrated inJNK1 ASO-transfected hepatocytes. Taken together, these data demonstratethat decreased BW or BW gain and lowered adiposity in the ASO-treatedmice were attributable to increased fuel combustion/metabolic rate anddecreased lipogenesis.

Antisense reduction of JNK1 activity lowers liver TG content andimproves hepatic steatosis. Additionally, plasma cholesterol levels areimproved. These changes were accompanied by increased expression ofhepatic UCP2 and PPARα genes and decreased expression of the key hepaticlipogenic genes including ACL, ACC1 and FAS. Without being bound to anytheory, these changes in gene expression indicate an increased shuntingof citrate into the TCA cycle and electron transport chain for oxidationand a reduced breakdown of it (by ACL) to produce acetyl-CoA forcholesterol and fatty acid synthesis. Improved hepatic steatosis andplasma cholesterol levels in JNK1 ASO-treated mice can therefore be dueto increased hepatic substrate oxidation and decreased hepaticlipogenesis. In vitro studies that showed decreased de novo sterolsynthesis and fatty acid synthesis and increased fatty acid oxidation inJNK1 ASO-transfected hepatocytes provide additional support. Inaddition, decreased expression of hepatic ApoB100 in JNK1 ASO-treatedmice was found. Reduction of hepatic ApoB100 expression lowers plasmacholesterol levels in different models of hyperlipidemia due to reducedhepatic cholesterol synthesis and export.

Specific reduction of JNK1 expression with ASO in just liver and fatprofoundly improved insulin sensitivity; normalized plasma glucose andinsulin levels and reduced glucose excursion during ITT and GTT. Thepositive effects were found to be accompanied by increased expression ofhepatic GK and GS and decreased expression of hepatic G6Pase and PKCs,and reduced expression of RBP4 in WAT. GK is the rate-limiting enzymefor hepatic synthesis of glucose-6-phosphate (which is further used forglycolysis or glycogen synthesis) from glucose that is taken-up fromblood, whereas G6Pase is the final “gate” for hepatic glucose output bybreaking down glucose-6-phosphate (that is from either gluconeogenesisor glycogenolysis) to release glucose into blood. GS is therate-limiting enzyme for glycogen synthesis that usesglucose-6-phosphate as the primary substrate. These changes in geneexpression indicate that antisense reduction of JNK1 expression improvesliver and even whole body insulin sensitivity, promotes blood glucoseutilization and/or storage in liver while inhibiting hepatic glucoseoutput, thus, resulting in improved blood glucose and insulin levels. Animproved insulin signaling activity in JNK1 ASO treated mice showingdecreased phosphorylation of IRS1^(Ser302/307) and increasedphosphorylation of Akt^(Ser473) in response to insulin furtherdemonstrates that antisense reduction of JNK1 expression improvesinsulin sensitivity.

Obesity, which is tightly associated with type 2 diabetes,hyperlipidemia, and fatty liver diseases, has become epidemic worldwide.JNK1 plays an important role in metabolism and energy homeostasis andantisense reduction of its expression in liver and fat increasesmetabolic rate and improves body weight and adiposity, which isaccompanied by improved liver steatosis, hypercholesterolemia andinsulin sensitivity in both genetically leptin-deficient anddiet-induced obese mice. Therefore, JNK1 is a useful therapeutic targetfor the treatment of obesity and related metabolic disorders.

The antisense compounds provided herein are therefore useful fortreating a number of metabolic conditions, including diabetes, obesityand metabolic syndrome. Such treatments encompass a therapeutic regimenthat results in a clinically desirable outcome. The clinically desiredoutcomes can be tied to glucose metabolism. For example, the antisensecompounds and methods provided herein are useful for improving bloodglucose control or tolerance and for improving insulin sensitivity in asubject in need thereof. The antisense compounds and methods providedherein are also useful for reducing plasma resistin levels in a subjectin need thereof. The antisense compounds and methods provided herein arealso useful for reducing glucose levels in a subject in need thereof.The compounds and methods are particularly useful for reducing blood,plasma and/or serum glucose levels. The compounds and methods are usefulfor reducing both fed and fasting glucose levels. Such clinical outcomesare desirable in disease and disorders related to glucose metabolism andinsulin resistance including, for example, diabetes, particularly typeII diabetes, obesity and metabolic syndrome. Therefore, the antisensecompounds and methods provided herein are useful for the treatment ofsuch diseases and disorders.

The clinically desirable outcomes can also be tied to lipid metabolism.For example, the antisense compounds and methods provided herein arealso useful for the reduction of lipids in a subject in need thereof,particularly serum lipids. The reduction in lipids can result from alowering of lipogenisis and particularly a lowering of lipogenic genesincluding, but not limited to ACC1, ACC2, FAS, SCD1 and DGAT1. Inparticular, the antisense compounds and methods are useful for reducingtriglyceride and cholesterol levels in a subject in need thereof. Thecompounds and methods are particularly useful for reducing plasmatriglyceride levels and plasma cholesterol levels. The compounds andmethods are also particularly useful for reducing liver triglyceridelevels. Additionally, the antisense compounds and methods providedherein are also useful for improving liver steatosis. The compounds andmethods are also particularly useful for increasing metabolic rate and,in turn, lowering body weight gain. The compounds and methods are alsoparticularly useful for lowering epididymal fat pad weight and wholebody fat content. Such clinical outcomes are desirable in diseases anddisorders of lipid as well as glucose metabolism and insulin resistanceincluding, for example, diabetes, metabolic syndrome, obesity,hypercholesterolemia, mixed dyslipidemia, atherosclerosis, coronaryheart disease, diabetes, type II diabetes, type II diabetes withdyslipidemia, dyslipidemia, hypertriglyceridemia, hyperlipidemia,hyperfattyacidemia, hepatic steatosis, non-alcoholic steatohepatitis(NASH), or non-alcoholic fatty liver disease (NAFLD). NAFLD is acondition characterized by fatty inflammation of the liver that is notdue to excessive alcohol use (for example, alcohol consumption of over20 g/day). In certain embodiments, NAFLD is related to insulinresistance and the metabolic syndrome. NASH is a condition characterizedby inflammation and the accumulation of fat and fibrous tissue in theliver that is not due to excessive alcohol use. NASH is an extreme formof NAFLD. Therefore, the antisense compounds and methods provided hereinare useful for the treatment of such diseases and disorders.

Elevated levels of blood glucose and triglycerides are recognized asmajor risk factors for development of diabetes, obesity and metabolicsyndrome. Elevated blood glucose levels or elevated triglyceride levelsare also considered a risk factor in the development and progression ofatherosclerosis. Atherosclerosis can lead to coronary heart disease,stroke, or peripheral vascular disease. Accordingly provided herein arepharmaceutical agents for lowering elevated levels of blood glucose andtriglycerides.

Metabolic syndrome is a condition characterized by a clustering of lipidand non-lipid cardiovascular risk factors of metabolic origin. Incertain embodiments, metabolic syndrome is identified by the presence ofany 3 of the following factors: waist circumference of greater than 102cm in men or greater than 88 cm in women; serum triglyceride of at least150 mg/dL; HDL-C less than 40 mg/dL in men or less than 50 mg/dL inwomen; blood pressure of at least 130/85 mmHg; and fasting glucose of atleast 110 mg/dL. These determinants can be readily measured in clinicalpractice (JAMA, 2001, 285: 2486-2497). Accordingly, the compounds andmethods provided herein can be used to treat individuals exhibiting oneor more risk factors for metabolic syndrome. Particularly, the compoundsand methods provided herein can be used to reduce body weight, therebylikely reducing waist circumference, and fasting glucose levels.

As illustrated herein, administration of an antisense oligonucleotidetargeted to JNK1 to animals models of diabetes and obesity which exhibitinsulin resistance, hyperglycemia and hyperlipidemia, resulted inantisense inhibition of JNK1, a reduction in plasma glucose andtriglyceride levels and reduction of liver triglycerides. Reduction intriglycerides was also accompanied by a reduction in lipogenic genes.Particularly, expression of ACC1, ACC2, FAS, SCD1 and DGAT1 werereduced. Thus, it is demonstrated that in an experimental model ofhyperglycemia and hyperlipidemia, antisense inhibition of JNK1 resultsin reduced glucose and triglyceride levels and reduced lipogenesis.Accordingly, provided herein are methods of reducing lipogenesis, bloodglucose and triglyceride levels through the administration of anantisense compound targeted to a JNK1 nucleic acid. Blood glucose andtriglyceride levels are considered a risk factor for development ofdiabetes, obesity and metabolic syndrome. Accordingly, also providedherein are methods for the treatment, prevention and/or amelioration ofdiabetes, obesity and metabolic syndrome, and for the treatment,prevention and/or amelioration of associated disorders. Also providedherein are methods for the treatment of conditions characterized byelevated liver triglycerides, such as hepatic steatosis.

Certain Indications

In certain embodiments, the invention provides methods of treating asubject comprising administering one or more pharmaceutical compositionsof the present invention. In certain embodiments, the individual hasdiabetes, obesity, metabolic syndrome and/or associated disordersincluding but not limited to hypercholesterolemia, mixed dyslipidemia,atherosclerosis, a risk of developing atherosclerosis, coronary heartdisease, a history of coronary heart disease, early onset coronary heartdisease, one or more risk factors for coronary heart disease, type IIdiabetes, type II diabetes with dyslipidemia, dyslipidemia,hypertriglyceridemia, hyperlipidemia, hyperfattyacidemia, hepaticsteatosis, non-alcoholic steatohepatitis, or non-alcoholic fatty liverdisease.

Hypercholesterolemia is a condition characterized by elevated serumcholesterol. Hyperlipidemia is a condition characterized by elevatedserum lipids. Hypertriglyceridemia is a condition characterized byelevated triglyceride levels. Non-familial hypercholesterolemia is acondition characterized by elevated cholesterol that is not the resultof a single gene mutation. Is polygenic hypercholesterolemia is acondition characterized by elevated cholesterol that results from theinfluence of a variety of genetic factors. In certain embodiments,polygenic hypercholesterolemia can be exacerbated by dietary intake oflipids. Familial hypercholesterolemia (FH) is an autosomal dominantmetabolic disorder characterized by a mutation in the LDL-receptor(LDL-R) gene, markedly elevated LDL-C and premature onset ofatherosclerosis. A diagnosis of familial hypercholesterolemia is madewhen a individual meets one or more of the following criteria: genetictesting confirming 2 mutated LDL-receptor genes; genetic testingconfirming one mutated LDL-receptor gene; document history of untreatedserum LDL-cholesterol greater than 500 mg/dL; tendinous and/or cutaneousxanthoma prior to age 10 years; or, both parents have documentedelevated serum LDL-cholesterol prior to lipid-lowering therapyconsistent with heterozygous familial hypercholesterolemia. Homozygousfamilial hypercholesterolemia (HoFH) is a condition characterized by amutation in both maternal and paternal LDL-R genes. Heterozygousfamilial hypercholesterolemia (HeFH) is a condition characterized by amutation in either the maternal or paternal LDL-R gene. Mixeddyslipidemia is a condition characterized by elevated serum cholesteroland elevated serum triglycerides. Diabetic dyslipidemia or Type IIdiabetes with dyslipidemia is a condition characterized by Type IIdiabetes, reduced HDL-C, elevated serum triglycerides, and elevatedsmall, dense LDL particles.

In one embodiment are methods for decreasing blood glucose levels ortriglyceride levels, or alternatively methods for treating obesity ormetabolic syndrome, by administering to a subject suffering fromelevated glucose or triglyceride levels a therapeutically effectiveamount of an antisense compound targeted to a JNK1 nucleic acid. Inanother embodiment, a method of decreasing blood glucose or triglyceridelevels comprises selecting a subject in need of a decrease in bloodglucose or triglyceride levels, and administering to the individual atherapeutically effective amount of an antisense compound targeted to aJNK1 nucleic acid. In a further embodiment, a method of reducing risk ofdevelopment of obesity and metabolic syndrome includes selecting asubject having elevated blood glucose or triglyceride levels and one ormore additional indicators risk of development of obesity or metabolicsyndrome, and administering to the individual a therapeuticallyeffective amount of an antisense compound targeted to a JNK1 nucleicacid.

In one embodiment, administration of a therapeutically effective amountof an antisense compound targeted a JNK1 nucleic acid is accompanied bymonitoring of glucose levels or triglyceride levels in the serum of asubject, to determine a subject's response to administration of theantisense compound. A subject's response to administration of theantisense compound is used by a physician to determine the amount andduration of therapeutic intervention.

Atherosclerosis can lead to coronary heart disease, stroke, orperipheral vascular disease. Elevated blood glucose levels or elevatedtriglyceride levels are considered a risk factor in the development andprogression of atherosclerosis. Accordingly, in one embodiment, atherapeutically effective amount of an antisense compound targeted to aJNK1 nucleic acid is administered to a subject having atherosclerosis.In a further embodiment a therapeutically effective amount of antisensecompound targeted to a JNK1 nucleic acid is administered to a subjectsusceptible to atherosclerosis. Atherosclerosis is assessed directlythrough routine imaging techniques such as, for example, ultrasoundimaging techniques that reveal carotid intimomedial thickness.Accordingly, treatment and/or prevention of atherosclerosis furtherinclude monitoring atherosclerosis through routine imaging techniques.In one embodiment, administration of an antisense compound targeted to aJNK1 nucleic acid leads to a lessening of the severity ofatherosclerosis, as indicated by, for example, a reduction of carotidintimomedial thickness in arteries.

Measurements of cholesterol, lipoproteins and triglycerides are obtainedusing serum or plasma collected from a subject. Methods of obtainingserum or plasma samples are routine, as are methods of preparation ofthe serum samples for analysis of cholesterol, triglycerides, and otherserum markers.

A physician can determine the need for therapeutic intervention forindividuals in cases where more or less aggressive blood glucose ortriglyceride-lowering therapy is needed. The practice of the methodsherein can be applied to any altered guidelines provided by the NCEP, orother entities that establish guidelines for physicians used in treatingany of the diseases or conditions listed herein, for determiningcoronary heart disease risk and diagnosing metabolic syndrome.

In one embodiment, administration of an antisense compound targeted aJNK1 nucleic acid is parenteral administration. Parenteraladministration can be intravenous or subcutaneous administration.Accordingly, in another embodiment, administration of an antisensecompound targeted to a JNK1 nucleic acid is intravenous or subcutaneousadministration. Administration can include multiple doses of anantisense compound targeted to a JNK1 nucleic acid.

In certain embodiments a pharmaceutical composition comprising anantisense compound targeted to JNK1 is for use in therapy. In certainembodiments, the therapy is the reduction of blood glucose, serumtriglyceride or liver triglyceride in a subject. In certain embodiments,the therapy is the treatment of hypercholesterolemia, mixeddyslipidemia, atherosclerosis, a risk of developing atherosclerosis,coronary heart disease, a history of coronary heart disease, early onsetcoronary heart disease, one or more risk factors for coronary heartdisease, type II diabetes, type II diabetes with dyslipidemia,dyslipidemia, hypertriglyceridemia, hyperlipidemia, hyperfattyacidemia,hepatic steatosis, non-alcoholic steatohepatitis, or non-alcoholic fattyliver disease. In additional embodiments, the therapy is the reductionof CHD risk. CHD risk equivalents refers to indicators of clinicalatherosclerotic disease that confer a high risk for coronary heartdisease, and include clinical coronary heart disease, symptomaticcarotid artery disease, peripheral arterial disease, and/or abdominalaortic aneurysm. In certain the therapy is prevention ofatherosclerosis. In certain embodiments, the therapy is the preventionof coronary heart disease.

In certain embodiments pharmaceutical composition comprising anantisense compound targeted to JNK1 is used for the preparation of amedicament for reduction of blood glucose, serum triglyceride or livertriglyceride. In certain embodiments pharmaceutical compositioncomprising an antisense compound targeted to JNK1 is used for thepreparation of a medicament for reducing coronary heart disease risk. Incertain embodiments an antisense compound targeted to JNK1 is used forthe preparation of a medicament for the treatment ofhypercholesterolemia, mixed dyslipidemia, atherosclerosis, a risk ofdeveloping atherosclerosis, coronary heart disease, a history ofcoronary heart disease, early onset coronary heart disease, one or morerisk factors for coronary heart disease, type II diabetes, type IIdiabetes with dyslipidemia, dyslipidemia, hypertriglyceridemia,hyperlipidemia, hyperfattyacidemia, hepatic steatosis, non-alcoholicsteatohepatitis, or non-alcoholic fatty liver disease.

Certain Combination Therapies

In certain embodiments, one or more pharmaceutical compositions of thepresent invention are co-administered with one or more otherpharmaceutical agents. In certain embodiments, such one or more otherpharmaceutical agents are designed to treat the same disease orcondition as the one or more pharmaceutical compositions of the presentinvention. In certain embodiments, such one or more other pharmaceuticalagents are designed to treat a different disease or condition as the oneor more pharmaceutical compositions of the present invention. In certainembodiments, such one or more other pharmaceutical agents are designedto treat an undesired effect of one or more pharmaceutical compositionsof the present invention. In certain embodiments, one or morepharmaceutical compositions of the present invention are co-administeredwith another pharmaceutical agent to treat an undesired effect of thatother pharmaceutical agent. In certain embodiments, one or morepharmaceutical compositions of the present invention and one or moreother pharmaceutical agents are administered at the same time. Incertain embodiments, one or more pharmaceutical compositions of thepresent invention and one or more other pharmaceutical agents areadministered at different times. In certain embodiments, one or morepharmaceutical compositions of the present invention and one or moreother pharmaceutical agents are prepared together in a singleformulation. In certain embodiments, one or more pharmaceuticalcompositions of the present invention and one or more otherpharmaceutical agents are prepared separately.

In certain embodiments, pharmaceutical agents that can beco-administered with a pharmaceutical composition comprising anantisense compound targeted to a JNK1 nucleic acid includeglucose-lowering agents and therapies. In some embodiments, theglucose-lowering agent is a PPAR agonist (gamma, dual, or pan), adipeptidyl peptidase (IV) inhibitor, a GLP-1 analog, insulin or aninsulin analog, an insulin secretagogue, a SGLT2 inhibitor, a humanamylin analog, a biguanide, an alpha-glucosidase inhibitor, ameglitinide, a thiazolidinedione, or a sulfonylurea.

In some embodiments, the glucose-lowering therapeutic is a GLP-1 analog.In some embodiments, the GLP-1 analog is exendin-4 or liraglutide.

In other embodiments, the glucose-lowering therapeutic is asulfonylurea. In some embodiments, the sulfonylurea is acetohexamide,chlorpropamide, tolbutamide, tolazamide, glimepiride, a glipizide, aglyburide, or a gliclazide.

In some embodiments, the glucose lowering drug is a biguanide. In someembodiments, the biguanide is metformin, and in some embodiments, bloodglucose levels are decreased without increased lactic acidosis ascompared to the lactic acidosis observed after treatment with metforminalone.

In some embodiments, the glucose lowering drug is a meglitinide. In someembodiments, the meglitinide is nateglinide or repaglinide.

In some embodiments, the glucose-lowering drug is a thiazolidinedione.In some embodiments, the thiazolidinedione is pioglitazone,rosiglitazone, or troglitazone. In some embodiments, blood glucoselevels are decreased without greater weight gain than observed withrosiglitazone treatment alone.

In some embodiments, the glucose-lowering drug is an alpha-glucosidaseinhibitor. In some embodiments, the alpha-glucosidase inhibitor isacarbose or miglitol.

In a certain embodiment, a co-administered glucose-lowering agent isISIS 113715.

In a certain embodiment, glucose-lowering therapy is therapeuticlifestyle change.

In certain such embodiments, the glucose-lowering agent is administeredprior to administration of a pharmaceutical composition of the presentinvention. In certain such embodiments, the glucose-lowering agent isadministered following administration of a pharmaceutical composition ofthe present invention. In certain such embodiments the glucose-loweringagent is administered at the same time as a pharmaceutical compositionof the present invention. In certain such embodiments the dose of aco-administered glucose-lowering agent is the same as the dose thatwould be administered if the glucose-lowering agent was administeredalone. In certain such embodiments the dose of a co-administeredglucose-lowering agent is lower than the dose that would be administeredif the glucose-lowering agent was administered alone. In certain suchembodiments the dose of a co-administered glucose-lowering agent isgreater than the dose that would be administered if the glucose-loweringagent was administered alone.

In certain embodiments, pharmaceutical agents that can beco-administered with a pharmaceutical composition comprising anantisense compound targeted to a JNK1 nucleic acid include anti-obesityagents. Such anti-obesity agents therapeutics can be administered asdescribed above for glucose lowering agents.

Further provided is a method of administering an antisense compoundtargeted to a JNK1 nucleic acid via injection and further includingadministering a topical steroid at the injection site.

In certain embodiments, pharmaceutical agents that can beco-administered with a pharmaceutical composition of the presentinvention include lipid-lowering agents. In certain such embodiments,pharmaceutical agents that can be co-administered with a pharmaceuticalcomposition of the present invention include, but are not limited toatorvastatin, simvastatin, rosuvastatin, and ezetimibe. In certain suchembodiments, the lipid-lowering agent is administered prior toadministration of a pharmaceutical composition of the present invention.In certain such embodiments, the lipid-lowering agent is administeredfollowing administration of a pharmaceutical composition of the presentinvention. In certain such embodiments the lipid-lowering agent isadministered at the same time as a pharmaceutical composition of thepresent invention. In certain such embodiments the dose of aco-administered lipid-lowering agent is the same as the dose that wouldbe administered if the lipid-lowering agent was administered alone. Incertain such embodiments the dose of a co-administered lipid-loweringagent is lower than the dose that would be administered if thelipid-lowering agent was administered alone. In certain such embodimentsthe dose of a co-administered lipid-lowering agent is greater than thedose that would be administered if the lipid-lowering agent wasadministered alone.

In certain embodiments, a co-administered lipid-lowering agent is aHMG-CoA reductase inhibitor. In certain such embodiments the HMG-CoAreductase inhibitor is a statin. In certain such embodiments the statinis selected from atorvastatin, simvastatin, pravastatin, fluvastatin,and rosuvastatin.

In certain embodiments, a co-administered lipid-lowering agent is acholesterol absorption inhibitor. In certain such embodiments,cholesterol absorption inhibitor is ezetimibe.

In certain embodiments, a co-administered lipid-lowering agent is aco-formulated HMG-CoA reductase inhibitor and cholesterol absorptioninhibitor. In certain such embodiments the co-formulated lipid-loweringagent is ezetimibe/simvastatin.

In certain embodiments, a co-administered lipid-lowering agent is amicrosomal triglyceride transfer protein inhibitor (MTP inhibitor).

In certain embodiments, a co-administered lipid-lowering agent is anoligonucleotide targeted to ApoB.

In certain embodiments, a co-administered pharmaceutical agent is a bileacid sequestrant. In certain such embodiments, the bile acid sequestrantis selected from cholestyramine, colestipol, and colesevelam.

In certain embodiments, a co-administered pharmaceutical agent is anicotinic acid. In certain such embodiments, the nicotinic acid isselected from immediate release nicotinic acid, extended releasenicotinic acid, and sustained release nicotinic acid.

In certain embodiments, a co-administered pharmaceutical agent is afibric acid. In certain such embodiments, a fibric acid is selected fromgemfibrozil, fenofibrate, clofibrate, bezafibrate, and ciprofibrate.

Further examples of pharmaceutical agents that can be co-administeredwith a pharmaceutical composition of the present invention include, butare not limited to, corticosteroids, including but not limited toprednisone; immunoglobulins, including, but not limited to intravenousimmunoglobulin (IVIg); analgesics (e.g., acetaminophen);anti-inflammatory agents, including, but not limited to non-steroidalanti-inflammatory drugs (e.g., ibuprofen, COX-1 inhibitors, and COX-2,inhibitors); salicylates; antibiotics; antivirals; antifungal agents;antidiabetic agents (e.g., biguanides, glucosidase inhibitors, insulins,sulfonylureas, and thiazolidenediones); adrenergic modifiers; diuretics;hormones (e.g., anabolic steroids, androgen, estrogen, calcitonin,progestin, somatostan, and thyroid hormones); immunomodulators; musclerelaxants; antihistamines; osteoporosis agents (e.g., biphosphonates,calcitonin, and estrogens); prostaglandins, antineoplastic agents;psychotherapeutic agents; sedatives; poison oak or poison sumacproducts; antibodies; and vaccines.

In certain embodiments, the pharmaceutical compositions of the presentinvention can be administered in conjunction with a lipid-loweringtherapy. In certain such embodiments, a lipid-lowering therapy istherapeutic lifestyle change. In certain such embodiments, alipid-lowering therapy is LDL apheresis.

In certain embodiments obesity is drug induced. In a particularembodiment obesity is induced by treatment with a psychotherapeutic drugor agent. Therapeutic use of certain psychothearapeutic agents, namelyatypical antipsychotic agents can increase the risk of metabolicabnormalities and there use is generally associated with weight gain andimpaired glucose tolerance. The percentage of patients gaining weightfollowing antipsychotic therapy can reach up to 80% depending on theantipsychotic used, with 30% or more developing obesity. Along withassociated medical complications, such metabolic abnormalities increasethe percentage of non-compliance patients and results in an increasedrisk of relapse.

Due to the ability of JNK1 antisense oligonucleotides to increasemetabolic rate and insulin sensitivity and reduce adiposity and weightgain, these compounds can be administered to reduce metabolicabnormalities associated with treatment with antipsychotic agents. Incertain embodiments the JNK1 antisense oligonucleotide is delivered in amethod of reducing metabolic abnormalities associated with thetherapeutic use of psychotherapeutic agents. Such weight inducingantipsychotic agents include, but are not limited to clozapine,olanzapine, aripiprazole, risperidone and ziprasidone.

In certain embodiments the JNK1 antisense oligonucleotide is deliveredconcomitant with delivery of the psychotherapeutic agent. Alternatively,delivery can be in the same formulation or can be administeredseparately. In a certain embodiment, the JNK1 antisense oligonucleotideis administered after treatment with an obesity inducing drug or agentis ceased. In a particular embodiment administering of the JNK1antisense compound results in increased metabolic rate or decreasingadiposity or both without affecting the CNS effects of thepsychotherapeutic agent.

In certain embodiments, JNK1 antisense oligonucleotides are administeredin combination either in the same formulation or separate formulationswith other anti-obesity drugs or agents. In certain embodiment, theanti-obesity agents are CNS based such as, but not limited to,sibutramine or GLP-1 based such as, but not limited to, liraglutide.

Antisense Compounds

Provided herein are antisense oligonucleotides that modulate the JNK1,JNK2 and JNK3 proteins. Such modulation is desirable for treating,alleviating or preventing various disorders or diseases, such as obesityand metabolic syndrome. Such inhibition is further desirable forpreventing or modulating the development of such diseases or disordersin an animal suspected of being, or known to be, prone to such diseasesor disorders.

Methods of modulating the expression of JNK1 proteins comprisingcontacting animals with oligonucleotides specifically hybridizable witha nucleic acid encoding a JNK1 protein are herein provided. Thesemethods are also useful for the diagnosis of conditions associated withsuch expression and activation.

Also provided herein are methods that comprise inhibiting JNK1-mediatedactivity using antisense oligonucleotides. These methods employ theoligonucleotides of the invention and are believed to be useful boththerapeutically and as clinical research and diagnostic tools. Providedare methods for inhibiting the expression of JNK1 from a nucleic acidfor the treatment, prevention or amelioration of a condition comprisingreducing body weight gain, reducing epididymal fat pad weight, reducingwhole body fat content, increasing metabolic rate, reducing fed plasmaglucose, reducing fasting plasma glucose, reducing fed plasma insulin,reducing fasted plasma insulin, improving glucose tolerance, improvinginsulin tolerance, improving liver steatosis, reducing plasmacholesterol, reducing plasma transaminase or a combination thereof.

Oligonucleotides are used herein in antisense modulation of the functionof DNA or messenger RNA (mRNA) encoding a protein the modulation ofwhich is desired, and ultimately to regulate the amount of such aprotein. Hybridization of an antisense oligonucleotide with its mRNAtarget interferes with the normal role of mRNA and causes a modulationof its function in cells. The functions of mRNA to be interfered withinclude all vital functions such as translocation of the RNA to the sitefor protein translation, actual translation of protein from the RNA,splicing of the RNA to yield one or more mRNA species, and possibly evenindependent catalytic activity which can be engaged in by the RNA. Theoverall effect of such interference with mRNA function is modulation ofthe expression of a protein, wherein modulation is either an increase(stimulation) or a decrease (inhibition) in the expression of theprotein. In the context of the present invention, inhibition is thepreferred form of modulation of gene expression.

It is preferred to target specific genes for antisense attack. Targetingan oligonucleotide to the associated nucleic acid, in the context ofthis invention, is a multistep process. The process begins with theidentification of a nucleic acid sequence whose function is to bemodulated. This can be, for example, a cellular gene (or mRNAtranscribed from the gene) whose expression is associated with aparticular disorder or disease state, or a foreign nucleic acid from aninfectious agent. In the present invention, the target is a cellulargene associated with hyperproliferative disorders. The targeting processalso includes determination of a site or sites within this gene for theoligonucleotide interaction to occur such that the desired effect,either detection or modulation of expression of the protein, willresult. Once the target site or sites have been identified,oligonucleotides are chosen which are sufficiently complementary to thetarget, i.e., hybridize sufficiently well and with sufficientspecificity to give the desired effect. Generally, there are fiveregions of a gene that can be targeted for antisense modulation: the 5′untranslated region (hereinafter, the “5′-UTR”), the translationinitiation codon region (hereinafter, the “tIR”), the open reading frame(hereinafter, the “ORF”), the translation termination codon region(hereinafter, the “tTR”) and the 3′ untranslated region (hereinafter,the “3′-UTR”). As is known in the art, these regions are arranged in atypical messenger RNA molecule in the following order (left to right, 5′to 3′): 5′-UTR, tIR, ORF, tTR, 3′-UTR. As is known in the art, althoughsome eukaryotic transcripts are directly translated, many ORFs containone or more sequences, known as “introns,” which are excised from atranscript before it is translated; the expressed (unexcised) portionsof the ORF are referred to as “exons” (Alberts et al., Molecular Biologyof the Cell, 1983, Garland Publishing Inc., New York, pp. 411-415).Furthermore, because many eukaryotic ORFs are a thousand nucleotides ormore in length, it is often convenient to subdivide the ORF into, e.g.,the 5′ ORF region, the central ORF region, and the 3′ ORF region. Insome instances, an ORF contains one or more sites that can be targeteddue to some functional significance in vivo. Examples of the lattertypes of sites include intragenic stem-loop structures (see, e.g., U.S.Pat. No. 5,512,438) and, in unprocessed mRNA molecules, intron/exonsplice sites.

Within the context of the present invention, one preferred intragenicsite is the region encompassing the translation initiation codon of theopen reading frame (ORF) of the gene. Because, as is known in the art,the translation initiation codon is typically 5′-AUG (in transcribedmRNA molecules; 5′-ATG in the corresponding DNA molecule), thetranslation initiation codon is also referred to as the “AUG codon,” the“start codon” or the “AUG start codon.” A minority of genes have atranslation initiation codon having the RNA sequence 5′-GUG, 5′-UUG or5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUG have been shown to function invivo. Furthermore, 5′-UUU functions as a translation initiation codon invitro (Brigstock et al., Growth Factors, 1990, 4, 45; Gelbert et al.,Somat. Cell. Mol. Genet., 1990, 16, 173; Gold and Stormo, in:Escherichia coli and Salmonella typhimurium: Cellular and MolecularBiology, Vol. 2, 1987, Neidhardt et al., Eds., American Society forMicrobiology, Washington, D.C., p. 1303). Thus, the terms “translationinitiation codon” and “start codon” can encompass many codon sequences,even though the initiator amino acid in each instance is typicallymethionine (in eukaryotes) or formylmethionine (prokaryotes). It is alsoknown in the art that eukaryotic and prokaryotic genes can have two ormore alternative start codons, any one of which can be preferentiallyutilized for translation initiation in a particular cell type or tissue,or under a particular set of conditions, in order to generate relatedpolypeptides having different amino terminal sequences (Markussen etal., Development, 1995, 121, 3723; Gao et al., Cancer Res., 1995, 55,743; McDermott et al., Gene, 1992, 117, 193; Perri et al., J. Biol.Chem., 1991, 266, 12536; French et al., J. Virol., 1989, 63, 3270;Pushpa-Rekha et al., J. Biol. Chem., 1995, 270, 26993; Monaco et al., J.Biol. Chem., 1994, 269, 347; DeVirgilio et al., Yeast, 1992, 8, 1043;Kanagasundaram et al., Biochim. Biophys. Acta, 1992, 1171, 198; Olsen etal., Mol. Endocrinol., 1991, 5, 1246; Saul et al., Appl. Environ.Microbiol., 1990, 56, 3117; Yaoita et al., Proc. Natl. Acad. Sci. USA,1990, 87, 7090; Rogers et al., EMBO J., 1990, 9, 2273). In the contextof the invention, “start codon” and “translation initiation codon” referto the codon or codons that are used in vivo to initiate translation ofan mRNA molecule transcribed from a gene encoding a JNK1 protein,regardless of the sequence(s) of such codons. It is also known in theart that a translation termination codon (or “stop codon”) of a gene canhave one of three sequences, i.e., 5′-UAA, 5′-UAG and 5′-UGA (thecorresponding DNA sequences are 5′-TAA, 5′-TAG and 5′-TGA,respectively). The terms “start codon region” and “translationinitiation region” refer to a portion of such an mRNA or gene thatencompasses from about 25 to about 50 contiguous nucleotides in eitherdirection (i.e., 5′ or 3′) from a translation initiation codon.Similarly, the terms “stop codon region” and “translation terminationregion” refer to a portion of such an mRNA or gene that encompasses fromabout 25 to about 50 contiguous nucleotides in either direction (i.e.,5′ or 3′) from a translation termination codon.

1. Oligonucleotides of the Invention: The present invention employsoligonucleotides for use in antisense modulation of one or more JNK1proteins. In the context of this invention, the term oligonucleotiderefers to an oligomer or polymer of ribonucleic acid or deoxyribonucleicacid. This term includes oligonucleotides composed ofnaturally-occurring nucleobases, sugars and covalent intersugar(backbone) linkages as well as oligonucleotides havingnon-naturally-occurring portions which function similarly. Such modifiedor substituted oligonucleotides are often preferred over native formsbecause of desirable properties such as, for example, enhanced cellularuptake, enhanced binding to target and increased stability in thepresence of nucleases.

An oligonucleotide is a polymer of a repeating unit generically known asa nucleotide. The oligonucleotides in accordance with this inventionpreferably comprise from about 8 to about 30 nucleotides. An unmodified(naturally occurring) nucleotide has three components: (1) anitrogen-containing heterocyclic base linked by one of its nitrogenatoms to (2) a 5-pentofuranosyl sugar and (3) a phosphate esterified toone of the 5′ or 3′ carbon atoms of the sugar. When incorporated into anoligonucleotide chain, the phosphate of a first nucleotide is alsoesterified to an adjacent sugar of a second, adjacent nucleotide via a3′-5′ phosphate linkage. The “backbone” of an unmodified oligonucleotideconsists of (2) and (3), that is, sugars linked together byphosphodiester linkages between the 5′ carbon of the sugar of a firstnucleotide and the 3′ carbon of a second, adjacent nucleotide. A“nucleoside” is the combination of (1) a nucleobase and (2) a sugar inthe absence of (3) a phosphate moiety (Kornberg, A., DNA Replication,W.H. Freeman & Co., San Francisco, 1980, pages 4-7). The backbone of anoligonucleotide positions a series of bases in a specific order; thewritten representation of this series of bases, which is conventionallywritten in 5′ to 3′ order, is known as a nucleotide sequence.

Oligonucleotides can comprise nucleotide sequences sufficient inidentity and number to effect specific hybridization with a particularnucleic acid. Such oligonucleotides which specifically hybridize to aportion of the sense strand of a gene are commonly described asantisense.” In the context of the invention, hybridization meanshydrogen bonding, which can be Watson-Crick, Hoogsteen or reversedHoogsteen hydrogen bonding, between complementary nucleotides. Forexample, adenine and thymine are complementary nucleobases which pairthrough the formation of hydrogen bonds. Complementary refers to thecapacity for precise pairing between two nucleotides. For example, if anucleotide at a certain position of an oligonucleotide is capable ofhydrogen bonding with a nucleotide at the same position of a DNA or RNAmolecule, then the oligonucleotide and the DNA or RNA are considered tobe complementary to each other at that position. The oligonucleotide andthe DNA or RNA are complementary to each other when a sufficient numberof corresponding positions in each molecule are occupied by nucleotideswhich can hydrogen bond with each other. An oligonucleotide isspecifically hybridizable to its target sequence due to the formation ofbase pairs between specific partner nucleobases in the interior of anucleic acid duplex. Among the naturally occurring nucleobases, guanine(G) binds to cytosine (C), and adenine (A) binds to thymine (T) oruracil (U). In addition to the equivalency of U (RNA) and T (DNA) aspartners for A, other naturally occurring nucleobase equivalents areknown, including 5-methylcytosine, 5-hydroxymethylcytosine (HMC),glycosyl HMC and gentiobiosyl HMC (C equivalents), and5-hydroxymethyluracil (U equivalent). Furthermore, synthetic nucleobaseswhich retain partner specificity are known in the art and include, forexample, 7-deaza-Guanine, which retains partner specificity for C. Thus,an oligonucleotide's capacity to specifically hybridize with its targetsequence will not be altered by any chemical modification to anucleobase in the nucleotide sequence of the oligonucleotide which doesnot significantly effect its specificity for the partner nucleobase inthe target oligonucleotide. It is understood in the art that anoligonucleotide need not be 100% complementary to its target DNAsequence to be specifically hybridizable. An oligonucleotide isspecifically hybridizable when there is a sufficient degree ofcomplementarity to avoid non-specific binding of the oligonucleotide tonon-target sequences under conditions in which specific binding isdesired, i.e., under physiological conditions in the case of in vivoassays or therapeutic treatment, and in the case of in vitro assays,under conditions in which the assays are performed.

Antisense oligonucleotides are commonly used as research reagents,diagnostic aids, and therapeutic agents. For example, antisenseoligonucleotides, which are able to inhibit gene expression withexquisite specificity, are often used by those of ordinary skill toelucidate the function of particular genes, for example to distinguishbetween the functions of various members of a biological pathway. Thisspecific inhibitory effect has, therefore, been harnessed by thoseskilled in the art for research uses. The specificity and sensitivity ofoligonucleotides is also harnessed by those of skill in the art fortherapeutic uses.

Modified Linkages: Specific examples of some preferred modifiedoligonucleotides envisioned for this invention include those containingphosphorothioates, phosphotriesters, methyl phosphonates, short chainalkyl or cycloalkyl intersugar linkages or short chain heteroatomic orheterocyclic intersugar linkages. Most preferred are oligonucleotideswith phosphorothioates and those with CH₂—NH—O—CH₂, CH₂—N(CH₃)—O—CH₂[known as a methylene(methylimino) or MMI backbone], CH₂—O—N(CH₃)—CH₂,CH₂—N(CH₃)—N(CH₃)—CH₂ and O—N(CH₃)—CH₂—CH₂ backbones, wherein the nativephosphodiester backbone is represented as O—P—O—CH₂). Also preferred areoligonucleotides having morpholino backbone structures (Summerton andWeller, U.S. Pat. No. 5,034,506). Further preferred are oligonucleotideswith NR—C(*)-CH₂—CH₂, CH₂—NR—C(*)-CH₂, CH₂—CH₂—NR—C(*), C(*)-NR—CH₂—CH₂and CH₂—C(*)-NR—CH₂ backbones, wherein “*” represents O or S (known asamide backbones; DeMesmaeker et al., WO 92/20823, published Nov. 26,1992). In other preferred embodiments, such as the peptide nucleic acid(PNA) backbone, the phosphodiester backbone of the oligonucleotide isreplaced with a polyamide backbone, the nucleobases being bound directlyor indirectly to the aza nitrogen atoms of the polyamide backbone(Nielsen et al., Science, 1991, 254, 1497; U.S. Pat. No. 5,539,082).

Modified Nucleobases The oligonucleotides of the invention canadditionally or alternatively include nucleobase modifications orsubstitutions. As used herein, “unmodified” or “natural” nucleobasesinclude adenine (A), guanine (G), thymine (T), cytosine (C) and uracil(U). Modified nucleobases include nucleobases found only infrequently ortransiently in natural nucleic acids, e.g., hypoxanthine,6-methyladenine, 5-methylcytosine, 5-hydroxymethylcytosine (HMC),glycosyl HMC and gentiobiosyl HMC, as well synthetic nucleobases, e.g.,2-aminoadenine, 2-thiouracil, 2-thiothymine, 5-bromouracil,5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine,N⁶(6-aminohexyl)adenine and 2,6-diaminopurine (Kornberg, A., DNAReplication, W.H. Freeman & Co., San Francisco, 1980, pages-75-77;Gebeyehu, G., et al., Nucleic Acids Res., 1987, 15, 4513).

Sugar Modifications: Modified oligonucleotides can also contain one ormore substituted sugar moieties. Preferred oligonucleotides comprise oneof the following at the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-,or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein thealkyl, alkenyl and alkynyl can be substituted or unsubstituted C₁ to C₁₀alkyl or C₂ to C₁₀ alkenyl and alkynyl. Particularly preferred areO[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃,O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from1 to about 10. Other preferred oligonucleotides comprise one of thefollowing at the 2′ position: C₁ to C₁₀ lower alkyl, substituted loweralkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br,CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving the pharmacokinetic properties of an oligonucleotide, or agroup for improving the pharmacodynamic properties of anoligonucleotide, and other substituents having similar properties. Apreferred modification includes an alkoxyalkoxy group, 2′-methoxyethoxy(2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martinet al., Helv. Chim. Acta, 1995, 78, 486-504). Further preferredmodifications include 2′-dimethylaminooxyethoxy, i.e., a2′-O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE and2′-dimethylaminoethoxyethoxy, i.e., 2′-O—CH₂—O—CH₂—N(CH₂)₂.

Other preferred modifications include 2′-methoxy (2′-O—CH₃),2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similarmodifications can also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′position of 5′ terminal nucleotide. Oligonucleotides can also have sugarmimetics such as cyclobutyl moieties in place of the pentofuranosylsugar. Representative United States patents that teach the preparationof such modified sugar structures include, but are not limited to, U.S.Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878;5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427;5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265;5,658,873; 5,670,633; and 5,700,920, each of which is hereinincorporated by reference.

Other Modifications: Similar modifications can also be made at otherpositions on the oligonucleotide, particularly the 3′ position of thesugar on the 3′ terminal nucleotide and the 5′ position of 5′ terminalnucleotide. The 5′ and 3′ termini of an oligonucleotide can also bemodified to serve as points of chemical conjugation of, e.g., lipophilicmoieties (see immediately subsequent paragraph), intercalating agents(Kuyavin et al., WO 96/32496, published Oct. 17, 1996; Nguyen et al.,U.S. Pat. No. 4,835,263, issued Can 30, 1989) or hydroxyalkyl groups(Helene et al., WO 96/34008, published Oct. 31, 1996).

Other positions within an oligonucleotide of the invention can be usedto chemically link thereto one or more effector groups to form anoligonucleotide conjugate. An “effector group” is a chemical moiety thatis capable of carrying out a particular chemical or biological function.Examples of such effector groups include, but are not limited to, an RNAcleaving group, a reporter group, an intercalator, a group for improvingthe pharmacokinetic properties of an oligonucleotide, or a group forimproving the pharmacodynamic properties of an oligonucleotide and othersubstituents having similar properties. A variety of chemical linkerscan be used to conjugate an effector group to an oligonucleotide of theinvention. As an example, U.S. Pat. No. 5,578,718 to Cook et al.discloses methods of attaching an alkylthio linker, which can be furtherderivatized to include additional groups, to ribofuranosyl positions,nucleosidic base positions, or on internucleoside linkages. Additionalmethods of conjugating oligonucleotides to various effector groups areknown in the art; see, e.g., Protocols for Oligonucleotide Conjugates(Methods in Molecular Biology, Volume 26) Agrawal, S., ed., HumanaPress, Totowa, N.J., 1994.

Another preferred additional or alternative modification of theoligonucleotides of the invention involves chemically linking to theoligonucleotide one or more lipophilic moieties which enhance thecellular uptake of the oligonucleotide. Such lipophilic moieties can belinked to an oligonucleotide at several different positions on theoligonucleotide. Some preferred positions include the 3′ position of thesugar of the 3′ terminal nucleotide, the 5′ position of the sugar of the5′ terminal nucleotide, and the 2′ position of the sugar of anynucleotide. The N⁶ position of a purine nucleobase can also be utilizedto link a lipophilic moiety to an oligonucleotide of the invention(Gebeyehu, G., et al., Nucleic Acids Res., 1987, 15, 4513). Suchlipophilic moieties include but are not limited to a cholesteryl moiety(Letsinger et al., Proc. Natl. Acad. Sci. U.S.A., 1989, 86, 6553),cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053),a thioether, e.g., hexyl-5-tritylthiol (Manoharan et al., Ann. N.Y.Acad. Sci., 1992, 660, 306; Manoharan et al., Bioorg. Med. Chem. Let.,1993, 3, 2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res.,1992, 20, 533), an aliphatic chain, e.g., dodecandiol or undecylresidues (Saison-Behmoaras et al., EMBO J., 1991, 10, 111; Kabanov etal., FEBS Lett., 1990, 259, 327; Svinarchuk et al., Biochimie, 1993, 75,49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651; Shea et al., Nucl. Acids Res., 1990,18, 3777), a polyamine or a polyethylene glycol chain (Manoharan et al.,Nucleosides & Nucleotides, 1995, 14, 969), or adamantane acetic acid(Manoharan et al., Tetrahedron Lett., 1995, 36, 3651), a palmityl moiety(Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229), or anoctadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke etal., J. Pharmacol. Exp. Ther., 1996, 277, 923). Oligonucleotidescomprising lipophilic moieties, and methods for preparing sucholigonucleotides, are disclosed in U.S. Pat. Nos. 5,138,045, 5,218,105and 5,459,255.

The present invention also includes oligonucleotides that aresubstantially chirally pure with regard to particular positions withinthe oligonucleotides. Examples of substantially chirally pureoligonucleotides include, but are not limited to, those havingphosphorothioate linkages that are at least 75% Sp or Rp (Cook et al.,U.S. Pat. No. 5,587,361) and those having substantially chirally pure(Sp or Rp) alkylphosphonate, phosphoamidate or phosphotriester linkages(Cook, U.S. Pat. Nos. 5,212,295 and 5,521,302).

Chimeric Oligonucleotides: The present invention also includesoligonucleotides which are chimeric oligonucleotides. “Chimeric”oligonucleotides or “chimeras,” in the context of this invention, areoligonucleotides which contain two or more chemically distinct regions,each made up of at least one nucleotide. These oligonucleotidestypically contain at least one region wherein the oligonucleotide ismodified so as to confer upon the oligonucleotide increased resistanceto nuclease degradation, increased cellular uptake, and/or increasedbinding affinity for the target nucleic acid. An additional region ofthe oligonucleotide can serve as a substrate for enzymes capable ofcleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is acellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex.Activation of RNase H, therefore, results in cleavage of the RNA target,thereby greatly enhancing the efficiency of antisense inhibition of geneexpression. Cleavage of the RNA target can be routinely detected by gelelectrophoresis and, if necessary, associated nucleic acid hybridizationtechniques known in the art. By way of example, such “chimeras” can be“gapmers,” i.e., oligonucleotides in which a central portion (the “gap”)of the oligonucleotide serves as a substrate for, e.g., RNase H, and the5′ and 3′ portions (the “wings”) are modified in such a fashion so as tohave greater affinity for the target RNA molecule but are unable tosupport nuclease activity (e.g., 2′-fluoro- or2′-methoxyethoxy-substituted). Other chimeras include “wingmers,” thatis, oligonucleotides in which the 5′ portion of the oligonucleotideserves as a substrate for, e.g., RNase H, whereas the 3′ portion ismodified in such a fashion so as to have greater affinity for the targetRNA molecule but is unable to support nuclease activity (e.g.,2′-fluoro- or 2′-methoxyethoxy-substituted), or vice-versa.

Synthesis: The oligonucleotides used in accordance with this inventioncan be conveniently and routinely made through the well-known techniqueof solid phase synthesis. Equipment for such synthesis is sold byseveral vendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art canadditionally or alternatively be employed. It is also known to usesimilar techniques to prepare other oligonucleotides such as thephosphorothioates and alkylated derivatives.

Teachings regarding the synthesis of particular modifiedoligonucleotides can be found in the following U.S. patents or pendingpatent applications, each of which is commonly assigned with thisapplication: U.S. Pat. Nos. 5,138,045 and 5,218,105, drawn to polyamineconjugated oligonucleotides; U.S. Pat. No. 5,212,295, drawn to monomersfor the preparation of oligonucleotides having chiral phosphoruslinkages; U.S. Pat. Nos. 5,378,825 and 5,541,307, drawn tooligonucleotides having modified backbones; U.S. Pat. No. 5,386,023,drawn to backbone modified oligonucleotides and the preparation thereofthrough reductive coupling; U.S. Pat. No. 5,457,191, drawn to modifiednucleobases based on the 3-deazapurine ring system and methods ofsynthesis thereof; U.S. Pat. No. 5,459,255, drawn to modifiednucleobases based on N-2 substituted purines; U.S. Pat. No. 5,521,302,drawn to processes for preparing oligonucleotides having chiralphosphorus linkages; U.S. Pat. No. 5,539,082, drawn to peptide nucleicacids; U.S. Pat. No. 5,554,746, drawn to oligonucleotides havingβ-lactam backbones; U.S. Pat. No. 5,571,902, drawn to methods andmaterials for the synthesis of oligonucleotides; U.S. Pat. No.5,578,718, drawn to nucleosides having alkylthio groups, wherein suchgroups can be used as linkers to other moieties attached at any of avariety of positions of the nucleoside; U.S. Pat. Nos. 5,587,361 and5,599,797, drawn to oligonucleotides having phosphorothioate linkages ofhigh chiral purity; U.S. Pat. No. 5,506,351, drawn to processes for thepreparation of 2′-O-alkyl guanosine and related compounds, including2,6-diaminopurine compounds; U.S. Pat. No. 5,587,469, drawn tooligonucleotides having N-2 substituted purines; U.S. Pat. No.5,587,470, drawn to oligonucleotides having 3-deazapurines; U.S. Pat.No. 5,223,168, issued Jun. 29, 1993, and U.S. Pat. No. 5,608,046, bothdrawn to conjugated 4′-desmethyl nucleoside analogs; U.S. Pat. No.5,602,240, and 5,610,289, drawn to backbone modified oligonucleotideanalogs; and U.S. patent application Ser. No. 08/383,666, filed Feb. 3,1995, and U.S. Pat. No. 5,459,255, drawn to, inter alia, methods ofsynthesizing 2′-fluoro-oligonucleotides.

5-methyl-cytosine: In 2′-methoxyethoxy-modified oligonucleotides,5-methyl-2′-methoxyethoxy-cytosine residues are used and are prepared asfollows. 2,2′-Anhydro[1-(β-D-arabinofuranosyl)-5-methyluridine]:5-Methyluridine (ribosylthymine, commercially available through Yamasa,Choshi, Japan) (72.0 g, 0.279 M), diphenylcarbonate (90.0 g, 0.420 M)and sodium bicarbonate (2.0 g, 0.024 M) were added to DMF (300 mL). Themixture was heated to reflux, with stirring, allowing the evolved carbondioxide gas to be released in a controlled manner. After 1 hour, theslightly darkened solution was concentrated under reduced pressure. Theresulting syrup was poured into diethylether (2.5 L), with stirring. Theproduct formed a gum. The ether was decanted and the residue wasdissolved in a minimum amount of methanol (ca. 400 mL). The solution waspoured into fresh ether (2.5 L) to yield a stiff gum. The ether wasdecanted and the gum was dried in a vacuum oven (60?C at 1 mm Hg for 24h) to give a solid which was crushed to a light tan powder (57 g, 85%crude yield). The material was used as is for further reactions.2′-O-Methoxyethyl-5-methyluridine: 2,2′-Anhydro-5-methyluridine (195 g,0.81 M), tris(2-methoxyethyl)borate (231 g, 0.98 M) and 2-methoxyethanol(1.2 L) were added to a 2 L stainless steel pressure vessel and placedin a pre-heated oil bath at 160?C. After heating for 48 hours at155-160?C, the vessel was opened and the solution evaporated to drynessand triturated with MeOH (200 mL). The residue was suspended in hotacetone (1 L). The insoluble salts were filtered, washed with acetone(150 mL) and the filtrate evaporated. The residue (280 g) was dissolvedin CH₃CN (600 mL) and evaporated. A silica gel column (3 kg) was packedin CH₂Cl₂/acetone/MeOH (20:5:3) containing 0.5% Et₃NH. The residue wasdissolved in CH₂Cl₂ (250 mL) and adsorbed onto silica (150 g) prior toloading onto the column. The product was eluted with the packing solventto give 160 g (63%) of product. 2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine: 2′-O-Methoxyethyl-5-methyluridine (160g, 0.506 M) was co-evaporated with pyridine (250 mL) and the driedresidue dissolved in pyridine (1.3 L). A first aliquot ofdimethoxytrityl chloride (94.3 g, 0.278 M) was added and the mixturestirred at room temperature for one hour. A second aliquot ofdimethoxytrityl chloride (94.3 g, 0.278 M) was added and the reactionstirred for an additional one hour. Methanol (170 mL) was then added tostop the reaction. HPLC showed the presence of approximately 70%product. The solvent was evaporated and triturated with CH₃CN (200 mL).The residue was dissolved in CHCl₃ (1.5 L) and extracted with 2×500 mLof saturated NaHCO₃ and 2×500 mL of saturated NaCl. The organic phasewas dried over Na₂SO₄, filtered and evaporated. 275 g of residue wasobtained. The residue was purified on a 3.5 kg silica gel column, packedand eluted with EtOAc/Hexane/Acetone (5:5:1) containing 0.5% Et₃NH. Thepure fractions were evaporated to give 164 g of product. Approximately20 g additional was obtained from the impure fractions to give a totalyield of 183 g (57%).3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine:2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (106 g, 0.167 M),DMF/pyridine (750 mL of a 3:1 mixture prepared from 562 mL of DMF and188 mL of pyridine) and acetic anhydride (24.38 mL, 0.258 M) werecombined and stirred at room temperature for 24 hours. The reaction wasmonitored by tlc by first quenching the tlc sample with the addition ofMeOH. Upon completion of the reaction, as judged by tlc, MeOH (50 mL)was added and the mixture evaporated at 35?C. The residue was dissolvedin CHCl₃ (800 mL) and extracted with 2×200 mL of saturated sodiumbicarbonate and 2×200 mL of saturated NaCl. The water layers were backextracted with 200 mL of CHCl₃. The combined organics were dried withsodium sulfate and evaporated to give 122 g of residue (approximately90% product). The residue was purified on a 3.5 kg silica gel column andeluted using EtOAc/Hexane (4:1). Pure product fractions were evaporatedto yield 96 g (84%). 3′-O-Acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine: A first solution wasprepared by dissolving 3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (96 g, 0.144 M) inCH₃CN (700 mL) and set aside. Triethylamine (189 mL, 1.44 M) was addedto a solution of triazole (90 g, 1.3 M) in CH₃CN (1 L), cooled to −5?Cand stirred for 0.5 h using an overhead stirrer. POCl₃ was addeddropwise, over a 30 minute period, to the stirred solution maintained at0-10?C, and the resulting mixture stirred for an additional 2 hours. Thefirst solution was added dropwise, over a 45 minute period, to the latersolution. The resulting reaction mixture was stored overnight in a coldroom. Salts were filtered from the reaction mixture and the solution wasevaporated. The residue was dissolved in EtOAc (1 L) and the insolublesolids were removed by filtration. The filtrate was washed with 1×300 mLof NaHCO₃ and 2×300 mL of saturated NaCl, dried over sodium sulfate andevaporated. The residue was triturated with EtOAc to give the titlecompound. 2′-O -Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine: Asolution of3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyl-4-triazoleuridine(103 g, 0.141 M) in dioxane (500 mL) and NH₄OH (30 mL) was stirred atroom temperature for 2 hours. The dioxane solution was evaporated andthe residue azeotroped with MeOH (2×200 mL). The residue was dissolvedin MeOH (300 mL) and transferred to a 2 liter stainless steel pressurevessel. Methanol (400 mL) saturated with NH₃ gas was added and thevessel heated to 100?C for 2 hours (thin layer chromatography, tlc,showed complete conversion). The vessel contents were evaporated todryness and the residue was dissolved in EtOAc (500 mL) and washed oncewith saturated NaCl (200 mL). The organics were dried over sodiumsulfate and the solvent was evaporated to give 85 g (95%) of the titlecompound.N⁴-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine:2′-O-Methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (85 g, 0.134 M)was dissolved in DMF (800 mL) and benzoic anhydride (37.2 g, 0.165 M)was added with stirring. After stirring for 3 hours, tlc showed thereaction to be approximately 95% complete. The solvent was evaporatedand the residue azeotroped with MeOH (200 mL). The residue was dissolvedin CHCl₃ (700 mL) and extracted with saturated NaHCO₃ (2×300 mL) andsaturated NaCl (2×300 mL), dried over MgSO₄ and evaporated to give aresidue (96 g). The residue was chromatographed on a 1.5 kg silicacolumn using EtOAc/Hexane (1:1) containing 0.5% Et₃NH as the elutingsolvent. The pure product fractions were evaporated to give 90 g (90%)of the title compound.N⁴-Benzoyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine-3′-amidite:N⁴-Benzoyl-2′-O -methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (74g, 0.10 M) was dissolved in CH₂Cl₂ (1 L). Tetrazole diisopropylamine(7.1 g) and 2-cyanoethoxy-tetra(isopropyl)phosphite (40.5 mL, 0.123 M)were added with stirring, under a nitrogen atmosphere. The resultingmixture was stirred for 20 hours at room temperature (tic showed thereaction to be 95% complete). The reaction mixture was extracted withsaturated NaHCO₃ (1× 300 mL) and saturated NaCl (3×300 mL). The aqueouswashes were back-extracted with CH₂Cl₂ (300 mL), and the extracts werecombined, dried over MgSO₄ and concentrated. The residue obtained waschromatographed on a 1.5 kg silica column using EtOAc\Hexane (3:1) asthe eluting solvent. The pure fractions were combined to give 90.6 g(87%) of the title compound. 2′-O-(Aminooxyethyl) nucleoside amiditesand 2′-O-(dimethylaminooxyethyl) nucleoside amidites2′-(Dimethylaminooxyethoxy) nucleoside amidites

2′-(Dimethylaminooxyethoxy) nucleoside amidites [also known in the artas 2′-O-(dimethylaminooxyethyl) nucleoside amidites] are prepared asdescribed in the following paragraphs. Adenosine, cytidine and guanosinenucleoside amidites are prepared similarly to the thymidine(5-methyluridine) except the exocyclic amines are protected with abenzoyl moiety in the case of adenosine and cytidine and with isobutyrylin the case of guanosine.5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine

O²-2′-anhydro-5-methyluridine (Pro. Bio. Sint., Varese, Italy, 100.0 g,0.416 mmol), dimethylaminopyridine (0.66 g, 0.013 eq, 0.0054 mmol) weredissolved in dry pyridine (500 ml) at ambient temperature under an argonatmosphere and with mechanical stirring. tert-Butyldiphenylchlorosilane(125.8 g, 119.0 mL, 1.1 eq, 0.458 mmol) was added in one portion. Thereaction was stirred for 16 h at ambient temperature. TLC (Rf 0.22,ethyl acetate) indicated a complete reaction. The solution wasconcentrated under reduced pressure to a thick oil. This was partitionedbetween dichloromethane (1 L) and saturated sodium bicarbonate (2×1 L)and brine (1 L). The organic layer was dried over sodium sulfate andconcentrated under reduced pressure to a thick oil. The oil wasdissolved in a 1:1 mixture of ethyl acetate and ethyl ether (600 mL) andthe solution was cooled to −10° C. The resulting crystalline product wascollected by filtration, washed with ethyl ether (3×200 mL) and dried(40° C., 1 mm Hg, 24 h) to 149 g (74.8%) of white solid. TLC and NMRwere consistent with pure product.5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine

In a 2 L stainless steel, unstirred pressure reactor was added borane intetrahydrofuran (1.0 M, 2.0 eq, 622 mL). In the fume hood and withmanual stirring, ethylene glycol (350 mL, excess) was added cautiouslyat first until the evolution of hydrogen gas subsided.5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine (149 g, 0.311mol) and sodium bicarbonate (0.074 g, 0.003 eq) were added with manualstirring. The reactor was sealed and heated in an oil bath until aninternal temperature of 160° C. was reached and then maintained for 16 h(pressure <100 psig). The reaction vessel was cooled to ambient andopened. TLC (Rf 0.67 for desired product and Rf 0.82 for ara-T sideproduct, ethyl acetate) indicated about 70% conversion to the product.In order to avoid additional side product formation, the reaction wasstopped, concentrated under reduced pressure (10 to 1 mm Hg) in a warmwater bath (40-100° C.) with the more extreme conditions used to removethe ethylene glycol. [Alternatively, once the low boiling solvent isgone, the remaining solution can be partitioned between ethyl acetateand water. The product will be in the organic phase.] The residue waspurified by column chromatography (2 kg silica gel, ethylacetate-hexanes gradient 1:1 to 4:1). The appropriate fractions werecombined, stripped and dried to product as a white crisp foam (84 g,50%), contaminated starting material (17.4 g) and pure reusable startingmaterial 20 g. The yield based on starting material less pure recoveredstarting material was 58%. TLC and NMR were consistent with 99% pureproduct.

2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine

5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine (20g, 36.98 mmol) was mixed with triphenylphosphine (11.63 g, 44.36 mmol)and N-hydroxyphthalimide (7.24 g, 44.36 mmol). It was then dried overP₂O₅ under high vacuum for two days at 40?C. The reaction mixture wasflushed with argon and dry THF (369.8 mL, Aldrich, sure seal bottle) wasadded to get a clear solution. Diethyl-azodicarboxylate (6.98 mL, 44.36mmol) was added dropwise to the reaction mixture. The rate of additionis maintained such that resulting deep red coloration is just dischargedbefore adding the next drop. After the addition was complete, thereaction was stirred for 4 hrs. By that time TLC showed the completionof the reaction (ethylacetate:hexane, 60:40). The solvent was evaporatedin vacuum. Residue obtained was placed on a flash column and eluted withethyl acetate:hexane (60:40), to get2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine aswhite foam (21.819 g, 86%).

5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine

2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine(3.1 g, 4.5 mmol) was dissolved in dry CH₂Cl₂ (4.5 mL) andmethylhydrazine (300 mL, 4.64 mmol) was added dropwise at −10?C to 0?C.After 1 h the mixture was filtered, the filtrate was washed with icecold CH₂Cl₂ and the combined organic phase was washed with water, brineand dried over anhydrous Na₂SO₄. The solution was concentrated to get2′-O-(aminooxyethyl) thymidine, which was then dissolved in MeOH (67.5mL). To this formaldehyde (20% aqueous solution, w/w, 1.1 eq.) was addedand the resulting mixture was stirred for 1 h. Solvent was removed undervacuum; residue chromatographed to get5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridineas white foam (1.95 g, 78%).

5′-O-tert-Butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine

5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine(1.77 g, 3.12 mmol) was dissolved in a solution of 1M pyridiniump-toluenesulfonate (PPTS) in dry MeOH (30.6 mL). Sodium cyanoborohydride(0.39 g, 6.13 mmol) was added to this solution at 10?C under inertatmosphere. The reaction mixture was stirred for 10 minutes at 10?C.After that the reaction vessel was removed from the ice bath and stirredat room temperature for 2 h, the reaction monitored by TLC (5% MeOH inCH₂Cl₂). Aqueous NaHCO₃ solution (5%, 10 mL) was added and extractedwith ethyl acetate (2×20 mL). Ethyl acetate phase was dried overanhydrous Na₂SO₄, evaporated to dryness. Residue was dissolved in asolution of 1M PPTS in MeOH (30.6 mL). Formaldehyde (20% w/w, 30 mL,3.37 mmol) was added and the reaction mixture was stirred at roomtemperature for 10 minutes. Reaction mixture cooled to 10?C in an icebath, sodium cyanoborohydride (0.39 g, 6.13 mmol) was added and reactionmixture stirred at 10?C for 10 minutes. After 10 minutes, the reactionmixture was removed from the ice bath and stirred at room temperaturefor 2 hrs. To the reaction mixture 5% NaHCO₃ (25 mL) solution was addedand extracted with ethyl acetate (2×25 mL). Ethyl acetate layer wasdried over anhydrous Na₂SO₄ and evaporated to dryness. The residueobtained was purified by flash column chromatography and eluted with 5%MeOH in CH₂Cl₂ to get5′-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridineas a white foam (14.6 g, 80%).

2′-O-(dimethylaminooxyethyl)-5-methyluridine

Triethylamine trihydrofluoride (3.91 mL, 24.0 mmol) was dissolved in dryTHF and triethylamine (1.67 mL, 12 mmol, dry, kept over KOH). Thismixture of triethylamine-2HF was then added to5-O-tert-butyldiphenylsilyl-2′-O-[N,N-dimethylaminooxyethyl]-5-methyluridine(1.40 g, 2.4 mmol) and stirred at room temperature for 24 hrs. Reactionwas monitored by TLC (5% MeOH in CH₂Cl₂). Solvent was removed undervacuum and the residue placed on a flash column and eluted with 10% MeOHin CH₂Cl₂ to get 2′-O -(dimethylaminooxyethyl)-5-methyluridine (766 mg,92.5%).

5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine

2′-O-(dimethylaminooxyethyl)-5-methyluridine (750 mg, 2.17 mmol) wasdried over P₂O₅ under high vacuum overnight at 40?C. It was thenco-evaporated with anhydrous pyridine (20 mL). The residue obtained wasdissolved in pyridine (11 mL) under argon atmosphere.4-dimethylaminopyridine (26.5 mg, 2.60 mmol), 4,4′-dimethoxytritylchloride (880 mg, 2.60 mmol) was added to the mixture and the reactionmixture was stirred at room temperature until all of the startingmaterial disappeared. Pyridine was removed under vacuum and the residuechromatographed and eluted with 10% MeOH in CH₂Cl₂ (containing a fewdrops of pyridine) to get5′-O-DMT-2′-O-(dimethylamino-oxyethyl)-5-methyluridine (1.13 g, 80%).

5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine (1.08 g, 1.67mmol) was co-evaporated with toluene (20 mL). To the residueN,N-diisopropylamine tetrazonide (0.29 g, 1.67 mmol) was added and driedover P₂O₅ under high vacuum overnight at 40?C. Then the reaction mixturewas dissolved in anhydrous acetonitrile (8.4 mL) and2-cyanoethyl-N,N,N¹,N¹-tetraisopropylphosphoramidite (2.12 mL, 6.08mmol) was added. The reaction mixture was stirred at ambient temperaturefor 4 hrs under inert atmosphere. The progress of the reaction wasmonitored by TLC (hexane:ethyl acetate 1:1). The solvent was evaporated,then the residue was dissolved in ethyl acetate (70 mL) and washed with5% aqueous NaHCO₃ (40 mL). Ethyl acetate layer was dried over anhydrousNa₂SO₄ and concentrated. Residue obtained was chromatographed (ethylacetate as eluent) to get5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]as a foam (1.04 g, 74.9%).

2′-(Aminooxyethoxy) nucleoside amidites

2′-(Aminooxyethoxy) nucleoside amidites [also known in the art as2′-O-(aminooxyethyl) nucleoside amidites] are prepared as described inthe following paragraphs. Adenosine, cytidine and thymidine nucleosideamidites are prepared similarly.

N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]

The 2′-O-aminooxyethyl guanosine analog can be obtained by selective2′-O-alkylation of diaminopurine riboside. Multigram quantities ofdiaminopurine riboside can be purchased from Schering AG (Berlin) toprovide 2′-O-(2-ethylacetyl) diaminopurine riboside along with a minoramount of the 3′-O -isomer. 2′-O-(2-ethylacetyl) diaminopurine ribosidecan be resolved and converted to 2′-O-(2-ethylacetyl)guanosine bytreatment with adenosine deaminase. (McGee, D. P. C., Cook, P. D.,Guinosso, C. J., WO 94/02501 A1 940203.) Standard protection proceduresshould afford 2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosineand2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosinewhich can be reduced to provide2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine.As before the hydroxyl group can be displaced by N-hydroxyphthalimidevia a Mitsunobu reaction, and the protected nucleoside canphosphitylated as usual to yield2-N-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite].

Bioequivalents: The compounds of the invention encompass anypharmaceutically acceptable salts, esters, or salts of such esters, orany other compound which, upon administration to an animal including ahuman, is capable of providing (directly or indirectly) the biologicallyactive metabolite or residue thereof. Accordingly, for example, thedisclosure is also drawn to “prodrugs” and “pharmaceutically acceptablesalts” of the oligonucleotides of the invention, pharmaceuticallyacceptable salts of such prodrugs, and other bioequivalents.

Oligonucleotide Prodrugs: The oligonucleotides of the invention canadditionally or alternatively be prepared to be delivered in a “prodrug”form. The term “prodrug” indicates a therapeutic agent that is preparedin an inactive form that is converted to an active form (i.e., drug)within the body or cells thereof by the action of endogenous enzymes orother chemicals and/or conditions. In particular, prodrug versions ofthe oligonucleotides of the invention are prepared as SATE[(S-acetyl-2-thioethyl)phosphate] derivatives according to the methodsdisclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993.

Pharmaceutically Acceptable Salts: The term A pharmaceuticallyacceptable salts” refers to physiologically and pharmaceuticallyacceptable salts of the oligonucleotides of the invention: i.e., saltsthat retain the desired biological activity of the parent compound anddo not impart undesired toxicological effects thereto.

Pharmaceutically acceptable base addition salts are formed with metalsor amines, such as alkali and alkaline earth metals or organic amines.Examples of metals used as cations are sodium, potassium, magnesium,calcium, and the like. Examples of suitable amines areN,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine(see, for example, Berge et al., “Pharmaceutical Salts,” J. of PharmaSci., 1977, 66, 1). The base addition salts of said acidic compounds areprepared by contacting the free acid form with a sufficient amount ofthe desired base to produce the salt in the conventional manner. Thefree acid form can be regenerated by contacting the salt form with anacid and isolating the free acid in the conventional manner. The freeacid forms differ from their respective salt forms somewhat in certainphysical properties such as solubility in polar solvents, but otherwisethe salts are equivalent to their respective free acid for purposes ofthe present invention. As used herein, a “pharmaceutical addition salt”includes a pharmaceutically acceptable salt of an acid form of one ofthe components of the compositions of the invention. These includeorganic or inorganic acid salts of the amines. Preferred acid salts arethe hydrochlorides, acetates, salicylates, nitrates and phosphates.Other suitable pharmaceutically acceptable salts are well known to thoseskilled in the art and include basic salts of a variety of inorganic andorganic acids, such as, for example, with inorganic acids, such as forexample hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoricacid; with organic carboxylic, sulfonic, sulfo or phospho acids orN-substituted sulfamic acids, for example acetic acid, propionic acid,glycolic acid, succinic acid, maleic acid, hydroxymaleic acid,methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid,oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid,benzoic acid, cinnamic acid, mandelic acid, salicylic acid,4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid,embonic acid, nicotinic acid or isonicotinic acid; and with amino acids,such as the 20 alpha-amino acids involved in the synthesis of proteinsin nature, for example glutamic acid or aspartic acid, and also withphenylacetic acid, methanesulfonic acid, ethanesulfonic acid,2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid,benzenesulfonic acid, 4-methylbenzenesulonic acid,naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (withthe formation of cyclamates), or with other acid organic compounds, suchas ascorbic acid. Pharmaceutically acceptable salts of compounds canalso be prepared with a pharmaceutically acceptable cation. Suitablepharmaceutically acceptable cations are well known to those skilled inthe art and include alkaline, alkaline earth, ammonium and quaternaryammonium cations. Carbonates or hydrogen carbonates are also possible.

For oligonucleotides, preferred examples of pharmaceutically acceptablesalts include but are not limited to (a) salts formed with cations suchas sodium, potassium, ammonium, magnesium, calcium, polyamines such asspermine and spermidine, etc.; (b) acid addition salts formed withinorganic acids, for example hydrochloric acid, hydrobromic acid,sulfuric acid, phosphoric acid, nitric acid and the like; (c) saltsformed with organic acids such as, for example, acetic acid, oxalicacid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconicacid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid,palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonicacid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (d)salts formed from elemental anions such as chlorine, bromine, andiodine.

Exemplary Utilities of the Invention: The oligonucleotides of thepresent invention specifically hybridize to nucleic acids (e.g., mRNAs)encoding a JNK1 protein. The oligonucleotides of the present inventioncan be utilized as therapeutic compounds, as diagnostic tools orresearch reagents that can be incorporated into kits, and inpurifications and cellular product preparations, as well as othermethodologies, which are appreciated by persons of ordinary skill in theart.

Assays and Diagnostic Applications: The oligonucleotides of the presentinvention can be used to detect the presence of JNK1 protein-specificnucleic acids in a cell or tissue sample. For example, radiolabeledoligonucleotides can be prepared by ³²P labeling at the 5′ end withpolynucleotide kinase. (Sambrook et al., Molecular Cloning. A LaboratoryManual, Cold Spring Harbor Laboratory Press, 1989, Volume 2, pg. 10.59.)Radiolabeled oligonucleotides are then contacted with cell or tissuesamples suspected of containing JNK1 protein message RNAs (and thus JNK1proteins), and the samples are washed to remove unbound oligonucleotide.Radioactivity remaining in the sample indicates the presence of boundoligonucleotide, which in turn indicates the presence of nucleic acidscomplementary to the oligonucleotide, and can be quantitated using ascintillation counter or other routine means. Expression of nucleicacids encoding these proteins is thus detected.

Radiolabeled oligonucleotides of the present invention can also be usedto perform autoradiography of tissues to determine the localization,distribution and quantitation of JNK1 proteins for research, diagnosticor therapeutic purposes. In such studies, tissue sections are treatedwith radiolabeled oligonucleotide and washed as described above, thenexposed to photographic emulsion according to routine autoradiographyprocedures. The emulsion, when developed, yields an image of silvergrains over the regions expressing a JNK1 protein gene. Quantitation ofthe silver grains permits detection of the expression of mRNA moleculesencoding these proteins and permits targeting of oligonucleotides tothese areas.

Analogous assays for fluorescent detection of expression of JNK1 proteinnucleic acids can be developed using oligonucleotides of the presentinvention which are conjugated with fluorescein or other fluorescenttags instead of radiolabeling. Such conjugations are routinelyaccomplished during solid phase synthesis using fluorescently-labeledamidites or controlled pore glass (CPG) columns. Fluorescein-labeledamidites and CPG are available from, e.g., Glen Research, Sterling Va.Other means of labeling oligonucleotides are known in the art (see,e.g., Ruth, Chapter 6 In Methods in Molecular Biology, Vol. 26:Protocols for Oligonucleotide Conjugates, Agrawal, ed., Humana PressInc., Totowa, N.J., 1994, pages 167-185).

Kits for detecting the presence or absence of expression of a JNK1protein can also be prepared. Such kits include an oligonucleotidetargeted to an appropriate gene, i.e., a gene encoding a JNK1 protein.Appropriate kit and assay formats, such as, e.g., “sandwich” assays, areknown in the art and can easily be adapted for use with theoligonucleotides of the invention. Hybridization of the oligonucleotidesof the invention with a nucleic acid encoding a JNK1 protein can bedetected by means known in the art. Such means can include conjugationof an enzyme to the oligonucleotide, radiolabelling of theoligonucleotide or any other suitable detection systems.

Protein Purifications: The oligonucleotides of the invention are alsouseful for the purification of specific Jun kinase proteins from cellsthat normally express a set of JNK proteins which are similar to eachother in terms of their polypeptide sequences and biochemicalproperties. As an example, the purification of a JNK1 protein from cellsthat expresses JNK1, JNK2 and JNK3 proteins can be enhanced by firsttreating such cells with oligonucleotides that inhibit the expression ofJNK2 and JNK3 and/or with oligonucleotides that increase the expressionof JNK1, because such treatments will increase the relative ratio ofJNK1 relative to JNK2 and JNK3. As a result, the yield of JNK1 fromsubsequent purification steps will be improved as the amount of thebiochemically similar (and thus likely to contaminate) JNK2 and JNK3proteins in extracts prepared from cells so treated will be diminished.

Biologically Active Oligonucleotides: The invention is also drawn to theadministration of oligonucleotides having biological activity tocultured cells, isolated tissues and organs and animals. By “havingbiological activity,” it is meant that the oligonucleotide functions tomodulate the expression of one or more genes in cultured cells, isolatedtissues or organs and/or animals. Such modulation can be achieved by anantisense oligonucleotide by a variety of mechanisms known in the art,including but not limited to transcriptional arrest; effects on RNAprocessing (capping, polyadenylation and splicing) and transportation;enhancement of cellular degradation of the target nucleic acid; andtranslational arrest (Crooke et al., Exp. Opin. Ther. Patents, 1996 6,855).

In an animal other than a human, the compositions and methods of theinvention can be used to study the function of one or more genes in theanimal. For example, antisense oligonucleotides have been systemicallyadministered to rats in order to study the role of theN-methyl-D-aspartate receptor in neuronal death, to mice in order toinvestigate the biological role of protein kinase C-a, and to rats inorder to examine the role of the neuropeptide Y1 receptor in anxiety(Wahlestedt et al., Nature, 1993, 363, 260; Dean et al., Proc. Natl.Acad. Sci. USA., 1994, 91, 11762; and Wahlestedt et al., Science, 1993,259, 528, respectively). In instances where complex families of relatedproteins are being investigated, “antisense knockouts” (i.e., inhibitionof a gene by systemic administration of antisense oligonucleotides) canrepresent the most accurate means for examining a specific member of thefamily (see, generally, Albert et al., Trends Pharmacol. Sci., 1994, 15,250).

The compositions and methods of the invention also have therapeutic usesin an animal, including a human, having (i.e., suffering from), or knownto be or suspected of being prone to having, a disease or disorder thatis treatable in whole or in part with one or more nucleic acids. Theterm “therapeutic uses” is intended to encompass prophylactic,palliative and curative uses wherein the oligonucleotides of theinvention are contacted with animal cells either in vivo or ex vivo.When contacted with animal cells ex vivo, a therapeutic use includesincorporating such cells into an animal after treatment with one or moreoligonucleotides of the invention.

For therapeutic uses, an animal suspected of having a disease ordisorder which can be treated or prevented by modulating the expressionor activity of a JNK1 protein is, for example, treated by administeringoligonucleotides in accordance with this invention. The oligonucleotidesof the invention can be utilized in pharmaceutical compositions byadding an effective amount of an oligonucleotide to a suitablepharmaceutically acceptable carrier such as, e.g., a diluent. Workers inthe field have identified antisense, triplex and other oligonucleotidecompositions which are capable of modulating expression of genesimplicated in viral, fungal and metabolic diseases. Antisenseoligonucleotides have been safely administered to humans and severalclinical trials are presently underway. It is thus established thatoligonucleotides can be useful therapeutic instrumentalities that can beconfigured to be useful in treatment regimes for treatment of cells,tissues and animals, especially humans. The following U.S. patentsdemonstrate palliative, therapeutic and other methods utilizingantisense oligonucleotides. U.S. Pat. No. 5,135,917 provides antisenseoligonucleotides that inhibit human interleukin-1 receptor expression.U.S. Pat. No. 5,098,890 is directed to antisense oligonucleotidescomplementary to the c-myb oncogene and antisense oligonucleotidetherapies for certain cancerous conditions. U.S. Pat. No. 5,087,617provides methods for treating cancer patients with antisenseoligonucleotides. U.S. Pat. No. 5,166,195 provides oligonucleotideinhibitors of Human Immunodeficiency Virus (HIV). U.S. Pat. No.5,004,810 provides oligomers capable of hybridizing to herpes simplexvirus Vmw65 mRNA and inhibiting replication. U.S. Pat. No. 5,194,428provides antisense oligonucleotides having antiviral activity againstinfluenza virus. U.S. Pat. No. 5,004,810 provides antisenseoligonucleotides and methods using them to inhibit HTLV-III replication.U.S. Pat. No. 5,286,717 provides oligonucleotides having a complementarybase sequence to a portion of an oncogene. U.S. Pat. No. 5,276,019 andU.S. Pat. No. 5,264,423 are directed to phosphorothioate oligonucleotideanalogs used to prevent replication of foreign nucleic acids in cells.U.S. Pat. No. 4,689,320 is directed to antisense oligonucleotides asantiviral agents specific to cytomegalovirus (CMV). U.S. Pat. No.5,098,890 provides oligonucleotides complementary to at least a portionof the mRNA transcript of the human c-myb gene. U.S. Pat. No. 5,242,906provides antisense oligonucleotides useful in the treatment of latentEpstein-Barr virus (EBV) infections.

As used herein, the term “disease, condition or disorder” includes anyabnormal condition of an organism or part that impairs normalphysiological functioning; isuch as obesity and metabolic syndrome. Asused herein, the term “prevention” means to delay or forestall onset ordevelopment of a condition or disease for a period of time from hours todays, preferably weeks to months. As used herein, the term“amelioration” means a lessening of at least one indicator of theseverity of a condition or disease. The severity of indicators can bedetermined by subjective or objective measures which are known to thoseskilled in the art. As used herein, “treatment” means to administer acomposition of the invention to effect an alteration or improvement ofthe disease or condition. Prevention, amelioration, and/or treatment canrequire administration of multiple doses at regular intervals, or priorto exposure to an agent (e.g., an allergen) to alter the course of thecondition or disease. Moreover, a single agent can be used in a singleindividual for each prevention, amelioration, and treatment of acondition or disease sequentially, or concurrently. The term “a diseaseor disorder that is treatable in whole or in part with one or morenucleic acids” refers to a disease or disorder, as herein defined, themanagement, modulation or treatment thereof, and/or therapeutic,curative, palliative and/or prophylactic relief therefrom, can beprovided via the administration of an antisense oligonucleotide.

Pharmaceutical Compositions: The formulation of pharmaceuticalcompositions comprising the oligonucleotides of the invention, and theirsubsequent administration, are believed to be within the skill of thosein the art.

Therapeutic Considerations: In general, for therapeutic applications, apatient (i.e., an animal, including a human, having or predisposed to adisease or disorder) is administered one or more oligonucleotides, inaccordance with the invention in a pharmaceutically acceptable carrierin doses ranging from 0.01 g to 100 g per kg of body weight depending onthe age of the patient and the severity of the disorder or disease statebeing treated. Further, the treatment regimen can last for a period oftime which will vary depending upon the nature of the particular diseaseor disorder, its severity and the overall condition of the patient, andcan extend from once daily to once every 20 years. In the context of theinvention, the term “treatment regimen” is meant to encompasstherapeutic, palliative and prophylactic modalities. Followingtreatment, the patient is monitored for changes in his/her condition andfor alleviation of the symptoms of the disorder or disease state. Thedosage of the nucleic acid can either be increased in the event thepatient does not respond significantly to current dosage levels, or thedose can be decreased if an alleviation of the symptoms of the disorderor disease state is observed, or if the disorder or disease state hasbeen ablated.

Dosing is dependent on severity and responsiveness of the disease stateto be treated, with the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution of thedisease state is achieved. Optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body of the patient.Persons of ordinary skill can easily determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages can vary dependingon the relative potency of individual oligonucleotides, and cangenerally be estimated based on EC₅₀s found to be effective in in vitroand in vivo animal models. In general, dosage is from 0.01 g to 100 gper kg of body weight, and can be given once or more daily, weekly,monthly or yearly, or even once every 2 to 20 years. An optimal dosingschedule is used to deliver a therapeutically effective amount of theoligonucleotide being administered via a particular mode ofadministration.

The term “therapeutically effective amount,” for the purposes of theinvention, refers to the amount of oligonucleotide-containingpharmaceutical composition which is effective to achieve an intendedpurpose without undesirable side effects (such as toxicity, irritationor allergic response). Although individual needs can vary, determinationof optimal ranges for effective amounts of pharmaceutical compositionsis within the skill of the art. Human doses can be extrapolated fromanimal studies (Katocs et al., Chapter 27 In: Remington's PharmaceuticalSciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa.,1990).

Generally, the dosage required to provide an effective amount of apharmaceutical composition, which can be adjusted by one skilled in theart, will vary depending on the age, health, physical condition, weight,type and extent of the disease or disorder of the recipient, frequencyof treatment, the nature of concurrent therapy (if any) and the natureand scope of the desired effect(s) (Nies et al., Chapter 3 In: Goodman &Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman etal., Eds., McGraw-Hill, New York, N.Y., 1996).

As used herein, the term “high risk individual” is meant to refer to asubject for whom it has been determined, via, e.g., individual or familyhistory or genetic testing, has a significantly higher than normalprobability of being susceptible to the onset or recurrence of a diseaseor disorder. As art of treatment regimen for a high risk individual, theindividual can be prophylactically treated to prevent the onset orrecurrence of the disease or disorder. The term “prophylacticallyeffective amount” is meant to refer to an amount of a pharmaceuticalcomposition which produces an effect observed as the prevention of theonset or recurrence of a disease or disorder. Prophylactically effectiveamounts of a pharmaceutical composition are typically determined by theeffect they have compared to the effect observed when a secondpharmaceutical composition lacking the active agent is administered to asimilarly situated individual.

Following successful treatment, it can be desirable to have the patientundergo maintenance therapy to prevent the recurrence of the diseasestate, wherein the nucleic acid is administered in maintenance doses,ranging from 0.01 g to 100 g per kg of body weight, once or more daily,to once every 20 years. For example, in the case of in individual knownor suspected of being prone to an autoimmune or inflammatory condition,prophylactic effects can be achieved by administration of preventativedoses, ranging from 0.01 μg to 100 g per kg of body weight, once or moredaily, to once every 20 years. In like fashion, a subject can be madeless susceptible to an inflammatory condition that is expected to occuras a result of some medical treatment, e.g., graft versus host diseaseresulting from the transplantation of cells, tissue or an organ into theindividual.

In some cases it can be more effective to treat a patient with anoligonucleotide of the invention in conjunction with other traditionaltherapeutic modalities in order to increase the efficacy of a treatmentregimen. A treatment regimen encompasses therapeutic, palliative andprophylactic modalities. For example, a patient can be treated withconventional chemotherapeutic agents, particularly those used for tumorand cancer treatment. Examples of such chemotherapeutic agents includebut are not limited to daunorubicin, daunomycin, dactinomycin,doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide,ifosfamide, cytosine arabinoside, bis-chloroethylnitrosurea, busulfan,mitomycin C, actinomycin D, mithramycin, prednisone,hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine,hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine,chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan,cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine (CA),5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine,vincristine, vinblastine, etoposide, trimetrexate, teniposide, cisplatinand diethylstilbestrol (DES). See, generally, The Merck Manual ofDiagnosis and Therapy, 15th Ed., pp. 1206-1228, Berkow et al., Eds.,Rahay, N.J., 1987). When used with the compounds of the invention, suchchemotherapeutic agents can be used individually (e.g., 5-FU andoligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for aperiod of time followed by MTX and oligonucleotide), or in combinationwith one or more other such chemotherapeutic agents (e.g., 5-FU, MTX andoligonucleotide, or 5-FU, radiotherapy and oligonucleotide).

In another preferred embodiment of the invention, a first antisenseoligonucleotide targeted to a first JNK1 protein is used in combinationwith a second antisense oligonucleotide targeted to a second JNK proteinin order to such JNK proteins to a more extensive degree than can beachieved when either oligonucleotide is used individually. In variousembodiments of the invention, the first and second JNK proteins whichare targeted by such oligonucleotides are identical, are different JNKproteins or are different isoforms of the same JNK protein.

Pharmaceutical Compositions Pharmaceutical compositions for thenon-parenteral administration of oligonucleotides can include sterileaqueous solutions which can also contain buffers, diluents and othersuitable additives. Pharmaceutically acceptable organic or inorganiccarrier substances suitable for non-parenteral administration which donot deleteriously react with oligonucleotides can be used. Suitablepharmaceutically acceptable carriers include, but are not limited to,water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose,amylose, magnesium stearate, talc, silicic acid, viscous paraffin,hydroxymethylcellulose, polyvinylpyrrolidone and the like. Thepharmaceutical compositions can be sterilized and, if desired, mixedwith auxiliary agents, e.g., lubricants, preservatives, stabilizers,wetting agents, emulsifiers, salts for influencing osmotic pressure,buffers, colorings flavorings and/or aromatic substances and the likewhich do not deleteriously react with the oligonucleotide(s) of thepharmaceutical composition. Pharmaceutical compositions in the form ofaqueous suspensions can contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. Optionally, such suspensions can also containstabilizers.

In one embodiment of the invention, an oligonucleotide is administeredvia the rectal mode. In particular, pharmaceutical compositions forrectal administration include foams, solutions (enemas) andsuppositories. Rectal suppositories for adults are usually tapered atone or both ends and typically weigh about 2 g each, with infant rectalsuppositories typically weighing about one-half as much, when the usualbase, cocoa butter, is used (Block, Chapter 87 In: Remington'sPharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co.,Easton, Pa., 1990).

In a preferred embodiment of the invention, one or more oligonucleotidesare administered via oral delivery. Pharmaceutical compositions for oraladministration include powders or granules, suspensions or solutions inwater or non-aqueous media, capsules, sachets, troches, tablets or SECs(soft elastic capsules or “caplets”). Thickeners, flavoring agents,diluents, emulsifiers, dispersing aids, carrier substances or binderscan be desirably added to such pharmaceutical compositions. The use ofsuch pharmaceutical compositions has the effect of delivering theoligonucleotide to the alimentary canal for exposure to the mucosathereof. Accordingly, the pharmaceutical composition can comprisematerial effective in protecting the oligonucleotide from pH extremes ofthe stomach, or in releasing the oligonucleotide over time, to optimizethe delivery thereof to a particular mucosal site. Enteric coatings foracid-resistant tablets, capsules and caplets are known in the art andtypically include acetate phthalate, propylene glycol and sorbitanmonoleate.

Various methods for producing pharmaceutical compositions for alimentarydelivery are well known in the art. See, generally, Nairn, Chapter 83;Block, Chapter 87; Rudnic et al., Chapter 89; Porter, Chapter 90; andLonger et al., Chapter 91 In: Remington's Pharmaceutical Sciences, 18thEd., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990. Theoligonucleotides of the invention can be incorporated in a known mannerinto customary pharmaceutical compositions, such as tablets, coatedtablets, pills, granules, aerosols, syrups, emulsions, suspensions andsolutions, using inert, non-toxic, pharmaceutically acceptable carriers(excipients). The therapeutically active compound should in each case bepresent here in a concentration of about 0.5% to about 95% by weight ofthe total mixture, i.e., in amounts which are sufficient to achieve thestated dosage range. The pharmaceutical compositions are prepared, forexample, by diluting the active compounds with pharmaceuticallyacceptable carriers, if appropriate using emulsifying agents and/ordispersing agents, and, for example, in the case where water is used asthe diluent, organic solvents can be used as auxiliary solvents ifappropriate. Pharmaceutical compositions can be formulated in aconventional manner using additional pharmaceutically acceptablecarriers as appropriate. Thus, the compositions can be prepared byconventional means with additional excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrates (e.g., starch or sodium starchglycolate); or wetting agents (e.g., sodium lauryl sulfate). Tablets canbe coated by methods well known in the art. The preparations can alsocontain flavoring, coloring and/or sweetening agents as appropriate.

The pharmaceutical compositions, which can conveniently be presented inunit dosage form, can be prepared according to conventional techniqueswell known in the pharmaceutical industry. Such techniques include thestep of bringing into association the active ingredient(s) with thepharmaceutically acceptable carrier(s). In general the pharmaceuticalcompositions are prepared by uniformly and intimately bringing intoassociation the active ingredient(s) with liquid excipients or finelydivided solid excipients or both, and then, if necessary, shaping theproduct.

Pharmaceutical compositions of the present invention suitable for oraladministration can be presented as discrete units such as capsules,cachets or tablets each containing predetermined amounts of the activeingredients; as powders or granules; as solutions or suspensions in anaqueous liquid or a non-aqueous liquid; or as oil-in-water emulsions orwater-in-oil liquid emulsions. A tablet can be made by compression ormolding, optionally with one or more accessory ingredients. Compressedtablets can be prepared by compressing in a suitable machine, the activeingredients in a free-flowing form such as a powder or granules,optionally mixed with a binder, lubricant, inert diluent, preservative,surface active or dispersing agent. Molded tablets can be made bymolding in a suitable machine a mixture of the powdered compoundmoistened with an inert liquid diluent. The tablets can optionally becoated or scored and can be formulated so as to provide slow orcontrolled release of the active ingredients therein. Pharmaceuticalcompositions for parenteral, intrathecal or intraventricularadministration, or colloidal dispersion systems, can include sterileaqueous solutions which can also contain buffers, diluents and othersuitable additives.

Penetration Enhancers: Pharmaceutical compositions comprising theoligonucleotides of the present invention can also include penetrationenhancers in order to enhance the alimentary delivery of theoligonucleotides. Penetration enhancers can be classified as belongingto one of five broad categories, i.e., fatty acids, bile salts,chelating agents, surfactants and non-surfactants (Lee et al., CriticalReviews in Therapeutic Drug Carrier Systems, 1991, 8, 91-192; Muranishi,Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7:1).

Fatty Acids: Various fatty acids and their derivatives which act aspenetration enhancers include, for example, oleic acid, lauric acid,capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid,linolenic acid, dicaprate, tricaprate, recinleate, monoolein (a.k.a.1-monooleoyl-rac-glycerol), caprylic acid, arichidonic acid, glyceryl1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, mono- and di-glycerides and physiologically acceptablesalts thereof (i.e., oleate, laurate, caprate, myristate, palmitate,stearate, linoleate, etc.) (Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems, 1990, 7, 1; El-Hariri et al., J.Pharm. Pharmacol., 1992, 44, 651).

Bile Salts: The physiological roles of bile include the facilitation ofdispersion and absorption of lipids and fat-soluble vitamins (Brunton,Chapter 38 In: Goodman & Gilman's The Pharmacological Basis ofTherapeutics, 9th Ed., Hardman et al., Eds., McGraw-Hill, New York,N.Y., 1996, pages 934-935). Various natural bile salts, and theirsynthetic derivatives, act as penetration enhancers. Thus, bile saltsinclude any of the naturally occurring components of bile as well as anyof their synthetic derivatives.

Chelating Agents: Chelating agents have the added advantage of alsoserving as DNase inhibitors and include, but are not limited to,disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates(e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acylderivatives of collagen, laureth-9 and N-amino acyl derivatives ofbeta-diketones (enamines)(Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems, 1990, 7, 1; Buur et al., J. ControlRel., 1990, 14, 43).

Surfactants: Surfactants include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether (Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92); and perfluorochemical emulsions, such as FC-43 (Takahashi et al.,J. Pharm. Phamacol., 1988, 40, 252).

Non-Surfactants: Non-surfactants include, for example, unsaturatedcyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page92); and non-steroidal anti-inflammatory agents such as diclofenacsodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm.Pharmacol., 1987, 39, 621).

Carrier Compounds: As used herein, “carrier compound” refers to anucleic acid, or analog thereof, which is inert (i.e., does not possessbiological activity per se) but is recognized as a nucleic acid by invivo processes that reduce the bioavailability of a nucleic acid havingbiological activity by, for example, degrading the biologically activenucleic acid or promoting its removal from circulation. Thecoadministration of a nucleic acid and a carrier compound, typicallywith an excess of the latter substance, can result in a substantialreduction of the amount of nucleic acid recovered in the liver, kidneyor other extracirculatory reservoirs, presumably due to competitionbetween the carrier compound and the nucleic acid for a common receptor.For example, the recovery of a partially phosphorothioatedoligonucleotide in hepatic tissue is reduced when it is coadministeredwith polyinosinic acid, dextran sulfate, polycytidic acid or4-acetamido-4′-isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al.,Antisense Res. Dev., 1995, 5, 115; Takakura et al., Antisense & Nucl.Acid Drug Dev., 1996, 6, 177).

Pharmaceutically Acceptable Carriers: In contrast to a carrier compound,a “pharmaceutically acceptable carrier” (excipient) is apharmaceutically acceptable solvent, suspending agent or any otherpharmacologically inert vehicle for delivering one or more nucleic acidsto an animal. The pharmaceutically acceptable carrier can be liquid orsolid and is selected with the planned manner of administration in mindso as to provide for the desired bulk, consistency, etc., when combinedwith a nucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutically acceptable carriers include, butare not limited to, binding agents (e.g., pregelatinised maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrates (e.g., starch, sodiumstarch glycolate, etc.); or wetting agents (e.g., sodium laurylsulphate, etc.). Sustained release oral delivery systems and/or entericcoatings for orally administered dosage forms are described in U.S. Pat.Nos. 4,704,295; 4,556,552; 4,309,406; and 4,309,404.

Miscellaneous Additional Components: The compositions of the presentinvention can additionally contain other adjunct componentsconventionally found in pharmaceutical compositions, at theirart-established usage levels. Thus, for example, the compositions cancontain additional compatible pharmaceutically-active materials such as,e.g., antipruritics, astringents, local anesthetics or anti-inflammatoryagents, or can contain additional materials useful in physicallyformulating various dosage forms of the composition of presentinvention, such as dyes, flavoring agents, preservatives, antioxidants,opacifiers, thickening agents and stabilizers. However, such materials,when added, should not unduly interfere with the biological activitiesof the components of the compositions of the invention.

Colloidal Dispersion Systems: Regardless of the method by which theoligonucleotides of the invention are introduced into a patient,colloidal dispersion systems can be used as delivery vehicles to enhancethe in vivo stability of the oligonucleotides and/or to target theoligonucleotides to a particular organ, tissue or cell type. Colloidaldispersion systems include, but are not limited to, macromoleculecomplexes, nanocapsules, microspheres, beads and lipid-based systemsincluding oil-in-water emulsions, micelles, mixed micelles andliposomes. A preferred colloidal dispersion system is a plurality ofliposomes, artificial membrane vesicles which can be used as cellulardelivery vehicles for bioactive agents in vitro and in vivo (Mannino etal., Biotechniques, 1988, 6, 682; Blume and Cevc, Biochem. et Biophys.Acta, 1990, 1029, 91; Lappalainen et al., Antiviral Res., 1994, 23, 119;Chonn and Cullis, Current Op. Biotech., 1995, 6, 698). It has been shownthat large unilamellar vesicles (LUV), which range in size from 0.2-0.4μm, can encapsulate a substantial percentage of an aqueous buffercontaining large macromolecules. RNA, DNA and intact virions can beencapsulated within the aqueous interior and delivered to brain cells ina biologically active form (Fraley et al., Trends Biochem. Sci., 1981,6, 77). The composition of the liposome is usually a combination oflipids, particularly phospholipids, in particular, high phase transitiontemperature phospholipids, usually in combination with one or moresteroids, particularly cholesterol. Examples of lipids useful inliposome production include phosphatidyl compounds, such asphosphatidylglycerol, phosphatidylcholine, phosphatidylserine,sphingolipids, phosphatidylethanolamine, cerebrosides and gangliosides.Particularly useful are diacyl phosphatidylglycerols, where the lipidmoiety contains from 14-18 carbon atoms, particularly from 16-18 carbonatoms, and is saturated (lacking double bonds within the 14-18 carbonatom chain). Illustrative phospholipids include phosphatidylcholine,dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.

The targeting of colloidal dispersion systems, including liposomes, canbe either passive or active. Passive targeting utilizes the naturaltendency of liposomes to distribute to cells of the reticuloendothelialsystem in organs that contain sinusoidal capillaries. Active targeting,by contrast, involves modification of the liposome by coupling thereto aspecific ligand such as a viral protein coat (Morishita et al., Proc.Natl. Acad. Sci. (U.S.A.), 1993, 90, 8474), monoclonal antibody (or asuitable binding portion thereof), sugar, glycolipid or protein (or asuitable oligopeptide fragment thereof), or by changing the compositionand/or size of the liposome in order to achieve distribution to organsand cell types other than the naturally occurring sites of localization.The surface of the targeted colloidal dispersion system can be modifiedin a variety of ways. In the case of a liposomal targeted deliverysystem, lipid groups can be incorporated into the lipid bilayer of theliposome in order to maintain the targeting ligand in close associationwith the lipid bilayer. Various linking groups can be used for joiningthe lipid chains to the targeting ligand. The targeting ligand, whichbinds a specific cell surface molecule found predominantly on cells towhich delivery of the oligonucleotides of the invention is desired, canbe, for example, (1) a hormone, growth factor or a suitable oligopeptidefragment thereof which is bound by a specific cellular receptorpredominantly expressed by cells to which delivery is desired or (2) apolyclonal or monoclonal antibody, or a suitable fragment thereof (e.g.,Fab; F(ab′)₂) which specifically binds an antigenic epitope foundpredominantly on targeted cells. Two or more bioactive agents (e.g., anoligonucleotide and a conventional drug; two oligonucleotides) can becombined within, and delivered by, a single liposome. It is alsopossible to add agents to colloidal dispersion systems which enhance theintercellular stability and/or targeting of the contents thereof.

Means of Administration: The present invention provides compositionscomprising oligonucleotides intended for administration to an animal.

Parenteral Delivery: The administration of an oligonucleotide of theinvention to an animal in a manner other than through the digestivecanal. Means of preparing and administering parenteral pharmaceuticalcompositions are known in the art (see, e.g., Avis, Chapter 84 In:Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., MackPublishing Co., Easton, Pa., 1990, pages 1545-1569). Parenteral means ofdelivery include, but are not limited to, the following illustrativeexamples.

Intravitreal injection, for the direct delivery of drug to the vitreoushumor of a mammalian eye, is described in U.S. Pat. No. 5,591,720, thecontents of which are hereby incorporated by reference. Means ofpreparing and administering ophthalmic preparations are known in the art(see, e.g., Mullins et al., Chapter 86 In: Remington's PharmaceuticalSciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa.,1990, pages 1581-1595).

Intravenous administration of antisense oligonucleotides to variousnon-human mammals has been described by Iversen (Chapter 26 In:Antisense Research and Applications, Crooke et al., Eds., CBC Press,Boca Raton, Fla., 1993, pages 461-469). Systemic delivery ofoligonucleotides to non-human mammals via intraperitoneal means has alsobeen described (Dean et al., Proc. Natl. Acad. Sci. (U.S.A.), 1994, 91,11766).

Intraluminal drug administration, for the direct delivery of drug to anisolated portion of a tubular organ or tissue (e.g., such as an artery,vein, ureter or urethra), can be desired for the treatment of patientswith diseases or conditions afflicting the lumen of such organs ortissues. To effect this mode of oligonucleotide administration, acatheter or cannula is surgically introduced by appropriate means. Forexample, for treatment of the left common carotid artery, a cannula isinserted thereinto via the external carotid artery. After isolation of aportion of the tubular organ or tissue for which treatment is sought, acomposition comprising the oligonucleotides of the invention is infusedthrough the cannula or catheter into the isolated segment. Afterincubation for from about 1 to about 120 minutes, during which theoligonucleotide is taken up by cells of the interior lumen of thevessel, the infusion cannula or catheter is removed and flow within thetubular organ or tissue is restored by removal of the ligatures whicheffected the isolation of a segment thereof (Morishita et al., Proc.Natl. Acad. Sci. U.S.A., 1993, 90, 8474). Antisense oligonucleotides canalso be combined with a biocompatible matrix, such as a hydrogelmaterial, and applied directly to vascular tissue in vivo (Rosenberg etal., U.S. Pat. No. 5,593,974, issued Jan. 14, 1997).

Intraventricular drug administration, for the direct delivery of drug tothe brain of a patient, can be desired for the treatment of patientswith diseases or conditions afflicting the brain. To effect this mode ofoligonucleotide administration, a silicon catheter is surgicallyintroduced into a ventricle of the brain of a human patient, and isconnected to a subcutaneous infusion pump (Medtronic Inc., Minneapolis,Minn.) that has been surgically implanted in the abdominal region (Zimmet al., Cancer Research, 1984, 44, 1698; Shaw, Cancer, 1993, 72(11Suppl.), 3416). The pump is used to inject the oligonucleotides andallows precise dosage adjustments and variation in dosage schedules withthe aid of an external programming device. The reservoir capacity of thepump is 18-20 mL and infusion rates can range from 0.1 mL/h to 1 mL/h.Depending on the frequency of administration, ranging from daily tomonthly, and the dose of drug to be administered, ranging from 0.01 μgto 100 g per kg of body weight, the pump reservoir can be refilled at3-10 week intervals. Refilling of the pump is accomplished bypercutaneous puncture of the self-sealing septum of the pump.

Intrathecal drug administration, for the introduction of a drug into thespinal column of a patient can be desired for the treatment of patientswith diseases of the central nervous system. To effect this route ofoligonucleotide administration, a silicon catheter is surgicallyimplanted into the L3-4 lumbar spinal interspace of a human patient, andis connected to a subcutaneous infusion pump which has been surgicallyimplanted in the upper abdominal region (Luer and Hatton, The Annals ofPharmacotherapy, 1993, 27, 912; Ettinger et al., Cancer, 1978, 41, 1270;Yaida et al., Regul. Pept., 1995, 59, 193). The pump is used to injectthe oligonucleotides and allows precise dosage adjustments andvariations in dose schedules with the aid of an external programmingdevice. The reservoir capacity of the pump is 18-20 mL, and infusionrates can vary from 0.1 mL/h to 1 mL/h. Depending on the frequency ofdrug administration, ranging from daily to monthly, and dosage of drugto be administered, ranging from 0.01 μg to 100 g per kg of body weight,the pump reservoir can be refilled at 3-10 week intervals. Refilling ofthe pump is accomplished by a single percutaneous puncture to theself-sealing septum of the pump. The distribution, stability andpharmacokinetics of oligonucleotides within the central nervous systemcan be followed according to known methods (Whitesell et al., Proc.Natl. Acad. Sci. (USA), 1993, 90, 4665).

To effect delivery of oligonucleotides to areas other than the brain orspinal column via this method, the silicon catheter is configured toconnect the subcutaneous infusion pump to, e.g., the hepatic artery, fordelivery to the liver (Kemeny et al., Cancer, 1993, 71, 1964). Infusionpumps can also be used to effect systemic delivery of oligonucleotides(Ewel et al., Cancer Research, 1992, 52, 3005; Rubenstein et al., J.Surg. Oncol., 1996, 62, 194).

Epidermal and Transdermal Delivery, in which pharmaceutical compositionscontaining drugs are applied topically, can be used to administer drugsto be absorbed by the local dermis or for further penetration andabsorption by underlying tissues, respectively. Means of preparing andadministering medications topically are known in the art (see, e.g.,Block, Chapter 87 In: Remington's Pharmaceutical Sciences, 18th Ed.,Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 1596-1609).

Vaginal Delivery provides local treatment and avoids first passmetabolism, degradation by digestive enzymes, and potential systemicside-effects. This mode of administration can be preferred for antisenseoligonucleotides targeted to pathogenic organisms for which the vaginais the usual habitat, e.g., Trichomonas vaginalis. In anotherembodiment, antisense oligonucleotides to genes encoding sperm-specificantibodies can be delivered by this mode of administration in order toincrease the probability of conception and subsequent pregnancy. Vaginalsuppositories (Block, Chapter 87 In: Remington's PharmaceuticalSciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa.,1990, pages 1609-1614) or topical ointments can be used to effect thismode of delivery.

Intravesical Delivery provides local treatment and avoids first passmetabolism, degradation by digestive enzymes, and potential systemicside-effects. However, the method requires urethral catheterization ofthe patient and a skilled staff. Nevertheless, this mode ofadministration can be preferred for antisense oligonucleotides targetedto pathogenic organisms, such as T. vaginalis, which can invade theurogenital tract.

Alimentary Delivery: The administration, directly or otherwise, to aportion of the alimentary canal of an animal. The term “alimentarycanal” refers to the tubular passage in an animal that functions in thedigestion and absorption of food and the elimination of food residue,which runs from the mouth to the anus, and any and all of its portionsor segments, e.g., the oral cavity, the esophagus, the stomach, thesmall and large intestines and the colon, as well as compound portionsthereof such as, e.g., the gastro-intestinal tract. Thus, the term“alimentary delivery” encompasses several routes of administrationincluding, but not limited to, oral, rectal, endoscopic andsublingual/buccal administration. A common requirement for these modesof administration is absorption over some portion or all of thealimentary tract and a need for efficient mucosal penetration of thenucleic acid(s) so administered.

Buccal/Sublingual Administration: Delivery of a drug via the oral mucosahas several desirable features, including, in many instances, a morerapid rise in plasma concentration of the drug than via oral delivery(Harvey, Chapter 35 In: Remington's Pharmaceutical Sciences, 18th Ed.,Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, page 711).Furthermore, because venous drainage from the mouth is to the superiorvena cava, this route also bypasses rapid first-pass metabolism by theliver. Both of these features contribute to the sublingual route beingthe mode of choice for nitroglycerin (Benet et al., Chapter 1 In:Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed.,Hardman et al., Eds., McGraw-Hill, New York, N.Y., 1996, page 7).

Endoscopic Administration: Endoscopy can be used for drug deliverydirectly to an interior portion of the alimentary tract. For example,endoscopic retrograde cystopancreatography (ERCP) takes advantage ofextended gastroscopy and permits selective access to the biliary tractand the pancreatic duct (Hirahata et al., Gan To Kagaku Ryoho, 1992,19(10 Suppl.), 1591). However, the procedure is unpleasant for thepatient, and requires a highly skilled staff.

Rectal Administration: Drugs administered by the oral route can often bealternatively administered by the lower enteral route, i.e., through theanal portal into the rectum or lower intestine. Rectal suppositories,retention enemas or rectal catheters can be used for this purpose andcan be preferred when patient compliance might otherwise be difficult toachieve (e.g., in pediatric and geriatric applications, or when thepatient is vomiting or unconscious). Rectal administration can result inmore prompt and higher blood levels than the oral route, but theconverse can be true as well (Harvey, Chapter 35 In: Remington'sPharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co.,Easton, Pa., 1990, page 711). Because about 50% of the drug that isabsorbed from the rectum will bypass the liver, administration by thisroute significantly reduces the potential for first-pass metabolism(Benet et al., Chapter 1 In: Goodman & Gilman's The PharmacologicalBasis of Therapeutics, 9th Ed., Hardman et al., Eds., McGraw-Hill, NewYork, N.Y., 1996).

Oral Administration: The preferred method of administration is oraldelivery, which is typically the most convenient route for access to thesystemic circulation. Absorption from the alimentary canal is governedby factors that are generally applicable, e.g., surface area forabsorption, blood flow to the site of absorption, the physical state ofthe drug and its concentration at the site of absorption (Benet et al.,Chapter 1 In: Goodman & Gilman's The Pharmacological Basis ofTherapeutics, 9th Ed., Hardman et al., Eds., McGraw-Hill, New York,N.Y., 1996, pages 5-7). A significant factor which can limit the oralbioavailability of a drug is the degree of “first pass effects.” Forexample, some substances have such a rapid hepatic uptake that only afraction of the material absorbed enters the peripheral blood (VanBerge-Henegouwen et al., Gastroenterology, 1977, 73, 300). Thecompositions and methods of the invention circumvent, at leastpartially, such first pass effects by providing improved uptake ofnucleic acids and thereby, e.g., causing the hepatic uptake system tobecome saturated and allowing a significant portion of the nucleic acidso administered to reach the peripheral circulation. Additionally oralternatively, the hepatic uptake system is saturated with one or moreinactive carrier compounds prior to administration of the active nucleicacid.

The following examples illustrate the invention and are not intended tolimit the same. Those skilled in the art will recognize, or be able toascertain through routine experimentation, numerous equivalents to thespecific substances and procedures described herein. Such equivalentsare considered to be within the scope of the present invention.

EXAMPLES Example 1 Synthesis of Oligonucleotides

General Synthetic Techniques: Oligonucleotides were synthesized on anautomated DNA synthesizer using standard phosphoramidite chemistry withoxidation using iodine. β-Cyanoethyldiisopropyl phosphoramidites werepurchased from Applied Biosystems (Foster City, Calif.). Forphosphorothioate oligonucleotides, the standard oxidation bottle wasreplaced by a 0.2 M solution of 3H-1,2-benzodithiole-3-one-1,1-dioxidein acetonitrile for the stepwise thiation of the phosphite linkages.

The synthesis of 2′-O-methyl- (a.k.a. 2′-methoxy-) phosphorothioateoligonucleotides is according to the procedures set forth abovesubstituting 2′-O-methyl β-cyanoethyldiisopropyl phosphoramidites(Chemgenes, Needham, Mass.) for standard phosphoramidites and increasingthe wait cycle after the pulse delivery of tetrazole and base to 360seconds.

Similarly, 2′-O-propyl- (a.k.a 2′-propoxy-) phosphorothioateoligonucleotides are prepared by slight modifications of this procedureand essentially according to procedures disclosed in U.S. patentapplication Ser. No. 08/383,666, filed Feb. 3, 1995, which is assignedto the same assignee as the instant application.

The 2′-fluoro-phosphorothioate oligonucleotides of the invention aresynthesized using 5′-dimethoxytrityl-3′-phosphoramidites and prepared asdisclosed in U.S. patent application Ser. No. 08/383,666, filed Feb. 3,1995, and U.S. Pat. No. 5,459,255, which issued Oct. 8, 1996, both ofwhich are assigned to the same assignee as the instant application. The2′-fluoro-oligonucleotides were prepared using phosphoramidite chemistryand a slight modification of the standard DNA synthesis protocol (i.e.,deprotection was effected using methanolic ammonia at room temperature).

The 2′-methoxyethoxy oligonucleotides were synthesized essentiallyaccording to the methods of Martin et al. (Helv. Chim. Acta, 1995, 78,486). For ease of synthesis, the 3′ nucleotide of the 2′-methoxyethoxyoligonucleotides was a deoxynucleotide, and 2′-O—CH₂CH₂OCH₃-cytosineswere 5-methyl cytosines, which were synthesized according to theprocedures described below.

PNA antisense analogs are prepared essentially as described in U.S. Pat.Nos. 5,539,082 and 5,539,083, both of which (1) issued Jul. 23, 1996,and (2) are assigned to the same assignee as the instant application.

Purification: After cleavage from the controlled pore glass column(Applied Biosystems) and deblocking in concentrated ammonium hydroxideat 55?C for 18 hours, the oligonucleotides were purified byprecipitation twice out of 0.5 M NaCl with 2.5 volumes ethanol.Analytical gel electrophoresis was accomplished in 20% acrylamide, 8 Murea, 45 mM Tris-borate buffer, pH 7.0. Oligodeoxynucleotides and theirphosphorothioate analogs were judged from electrophoresis to be greaterthan 80% full length material.

Example 2 Assays for Oligonucleotide-Mediated Inhibition of JNK mRNAExpression in Human Tumor Cells

In order to evaluate the activity of potential JNK-modulatingoligonucleotides, human lung carcinoma cell line A549 (American TypeCulture Collection, Rockville, Md. No. ATCC CCL-185) cells or other celllines as indicated in the Examples, were grown and treated witholigonucleotides or control solutions as detailed below. Afterharvesting, cellular extracts were prepared and examined for specificJNK mRNA levels or JNK protein levels (i.e., Northern or Western assays,respectively). In all cases, “% expression” refers to the amount ofJNK-specific signal in an oligonucleotide-treated cell relative to anuntreated cell (or a cell treated with a control solution that lacksoligonucleotide).

Northern Assays The mRNA expression of each JNK protein was determinedby using a nucleic acid probe specifically hybridizable thereto. Nucleicacid probes specific for JNK1, JNK2 and JNK3 are described in Examples3, 4 and 5, respectively. The probes were radiolabelled by means wellknown in the art (see, e.g., Short Protocols in Molecular Biology, 2ndEd., Ausubel et al., Eds., John Wiley & Sons, New York, 1992, pages 3-11to 2-3-44 and 4-17 to 4-18; Ruth, Chapter 6 In: Methods in MolecularBiology, Vol. 26: Protocols for Oligonucleotide Conjugates, Agrawal,ed., Humana Press Inc., Totowa, N.J., 1994, pages 167-185; and Chapter10 In: Molecular Cloning: A Laboratory Manual, 2nd Ed., Sambrook et al.,Eds., pages 10.1-10.70). The blots were stripped and reprobed with a³²P-labeled glyceraldehyde 3-phosphate dehydrogenase (G3PDH) probe(Clontech Laboratories, Inc., Palo Alto, Calif.) in order to confirmequal loading of RNA and to allow the levels of JNK transcripts to benormalized with regard to the G3PDH signals.

A549 cells were grown in T-75 flasks until 80-90% confluent. At thistime, the cells were washed twice with 10 mL of media (DMEM), followedby the addition of 5 mL of DMEM containing 20 ┘g/mL of LIPOFECTIN™(i.e., 1:1 (w/w) DOTMA/DOPE, Life Technologies, Gaithersburg, Md.;DOTMA=N-[1-(2,3-dioleyoxy)propyl]-N,N,N-trimethylammonium chloride;DOPE=dioleoyl phosphatidylethanolamine). The oligonucleotides were addedfrom a 10 μM stock solution to a final concentration of 400 nM, and thetwo solutions were mixed by swirling the flasks. As a control, cellswere treated with LIPOFECTIN™ without oligonucleotide under the sameconditions and for the same times as the oligonucleotide-treatedsamples. After 4 hours at 37° C., the medium was replaced with freshDMEM containing 10% serum. The cells were allowed to recover for 18hours. Total cellular RNA was then extracted in guanidinium, subject togel electrophoresis and transferred to a filter according to techniquesknown in the art (see, e.g., Chapter 7 In: Molecular Cloning: ALaboratory Manual, 2nd Ed., Sambrook et al., Eds., pages 7.1-7.87, andShort Protocols in Molecular Biology, 2nd Ed., Ausubel et al., Eds.,John Wiley & Sons, New York, 1992, pages 2-24 to 2-30 and 4-14 to 4-29).Filters were typically hybridized overnight to a probe specific for theparticular JNK gene of interest in hybridization buffer (25 mM KPO₄, pH7.4; 5×SSC; 5×Denhardt's solution, 100 g/ml Salmon sperm DNA and 50%formamide) (Alahari et al., Nucl. Acids Res., 1993, 21, 4079). This wasfollowed by two washes with 1×SSC, 0.1% SDS and two washes with0.25×SSC, 0.1% SDS. Hybridizing bands were visualized by exposure toX-OMAT AR film and quantitated using a PHOSPHORIMAGER™ essentiallyaccording to the manufacturer's instructions (Molecular Dynamics,Sunnyvale, Calif.).

Western Assays: A549 cells were grown and treated with oligonucleotidesas described above. Cells were lysed, and protein extracts wereelectrophoresed (SDS-PAGE) and transferred to nitrocellulose filters bymeans known in the art (see, e.g., Chapter 18 In: Molecular Cloning: ALaboratory Manual, 2nd Ed., Sambrook et al., Eds., pages 18.34,18.47-18.54 and 18.60-18.75)). The amount of each JNK protein wasdetermined by using a primary antibody that specifically recognizes theappropriate JNK protein. The primary antibodies specific for each JNKprotein are described in the appropriate Examples. The primaryantibodies were detected by means well known in the art (see, e.g.,Short Protocols in Molecular Biology, 2nd Ed., Ausubel et al., Eds.,John Wiley & Sons, New York, 1992, pages 10-33 to 10-35; and Chapter 18In: Molecular Cloning: A Laboratory Manual, 2nd Ed., Sambrook et al.,Eds., pages 18.1-18.75 and 18.86-18.88) and quantitated using aPHOSPHORIMAGER™ essentially according to the manufacturer's instructions(Molecular Dynamics, Sunnyvale, Calif.).

Levels of JNK proteins can also be quantitated by measuring the level oftheir corresponding kinase activity. Such kinase assays can be done ingels in situ (Hibi et al., Genes & Dev., 1993, 7, 2135) or afterimmunoprecipitation from cellular extracts (Derijard et al., Cell, 1994,76, 1025). Substrates and/or kits for such assays are commerciallyavailable from, for example, Upstate Biotechnology, Inc. (Lake Placid,N.Y.), New England Biolabs, Inc., (Beverly, Mass.) andCalbiochem-Novabiochem Biosciences, Inc., (La Jolla, Calif.).

Example 3 Oligonucleotide-Mediated Inhibition of JNK1 Expression

JNK1 oligonucleotide sequences: Table 1 lists the nucleotide sequencesof a set of oligonucleotides designed to specifically hybridize to JNK1mRNAs and their corresponding ISIS and SEQ ID numbers. The nucleotideco-ordinates of the target gene, JNK1, and gene target regions are alsoincluded. The nucleotide co-ordinates are derived from GenBank accessionNo. L26318 (SEQ ID NO: 87), locus name “HUMJNK1” (see also FIG. 1(A) ofDerijard et al., Cell, 1994, 76, 1025). The abbreviations for genetarget regions are as follows: 5′-UTR, 5′ untranslated region; tIR,translation initiation region; ORF, open reading frame; 3′-UTR, 3′untranslated region. The nucleotides of the oligonucleotides whosesequences are presented in Table 1 are connected by phosphorothioatelinkages and are unmodified at the 2′ position (i.e., 2′-deoxy). Itshould be noted that the oligonucleotide target co-ordinate positionsand gene target regions can vary within mRNAs encoding related isoformsof JNK1 (see subsection G, below).

In addition to hybridizing to human JNK1 mRNAs, the full oligonucleotidesequences of ISIS Nos. 12548 (SEQ ID NO: 17) and 12551 (SEQ ID NO: 20)hybridize to the 5′ ends of mRNAs from Rattus norvegicus that encode astress-activated protein kinase named “p54?” (Kyriakis et al., Nature,1994, 369, 156). Specifically, ISIS 12548 (SEQ ID NO: 17) hybridizes tobases 498-517 of GenBank accession No. L27129 (SEQ ID NO: 88), locusname “RATSAPKD,” and ISIS 12551 (SEQ ID NO: 20) hybridizes to bases803-822 of the same sequence.

JNK1-specific probes: In initial screenings of a set of oligonucleotidesderived from the JNK1 sequence (Table 2) for biological activity, a cDNAclone of JNK1 (Derijard et al., Cell, 1994, 76, 1025) was radiolabeledand used as a JNK1-specific probe in Northern blots. Alternatively,however, one or more of the oligonucleotides of Table 1 is detectablylabeled and used as a JNK1-specific probe.

TABLE 1 Nucleotide Sequences of JNK1 Oligonucleotides TARGET GENE GENEISIS NUCLEOTIDE SEQUENCE SEQ NUCLEOTIDE TARGET NO. (5′ -> 3′) ID NO:CO-ORDINATES REGION 11978 ATT-CTT-TCC-ACT-CTT-CTA-TT 1 1062-1081 ORF11979 CTC-CTC-CAA-GTC-CAT-AAC-TT 2 1094-1113 ORF 11980CCC-GTA-TAA-CTC-CAT-TCT-TG 3 1119-1138 ORF 11981CTG-TGC-TAA-AGG-AGA-GGG-CT 4 1142-1161 ORF 11982ATG-ATG-GAT-GCT-GAG-AGC-CA 5 1178-1197 3′-UTR 11983GTT-GAC-ATT-GAA-GAC-ACA-TC 6 1215-1234 3′-UTR 11984CTG-TAT-CAG-AGG-CCA-AAG-TC 7 1241-1260 3′-UTR 11985TGC-TGC-TTC-TAG-ACT-GCT-GT 8 1261-1280 3′-UTR 11986AGT-CAT-CTA-CAG-CAG-CCC-AG 9 1290-1309 3′-UTR 11987CCA-TCC-CTC-CCA-CCC-CCC-GA 10 1320-1339 3′-UTR 11988ATC-AAT-GAC-TAA-CCG-ACT-CC 11 1340-1359 3′-UTR 11989CAA-AAA-TAA-GAC-CAC-TGA-AT 12 1378-1397 3′-UTR 12463CAC-GCT-TGC-TTC-TGC-TCA-TG 13 0018-0037 tIR 12464CGG-CTT-AGC-TTC-TTG-ATT-GC 14 0175-0194 ORF 12538CCC-GCT-TGG-CAT-GAG-TCT-GA 15 0207-0226 ORF 12539CTC-TCT-GTA-GGC-CCG-CTT-GG 16 0218-0237 ORF 12548ATT-TGC-ATC-CAT-GAG-CTC-CA 17 0341-0360 ORF 12549CGT-TCC-TGC-AGT-CCT-GGC-CA 18 0533-0552 ORF 12550GGA-TGA-CCT-CGG-GTG-CTC-TG 19 0591-0610 ORF 12551CCC-ATA-ATG-CAC-CCC-ACA-GA 20 0646-0665 ORF 12552CGG-GTG-TTG-GAG-AGC-TTC-AT 21 0956-0975 ORF 12553TTT-GGT-GGT-GGA-GCT-TCT-GC 22 1006-1025 ORF 12554GGC-TGC-CCC-CGT-ATA-ACT-CC 23 1126-1145 ORF 12555TGC-TAA-AGG-AGA-GGG-CTG-CC 24 1139-1158 ORF 12556AGG-CCA-AAG-TCG-GAT-CTG-TT 25 1232-1251 3′-UTR 12557CCA-CCC-CCC-GAT-GGC-CCA-AG 26 1311-1330 3′-UTR

Activities of JNK1 oligonucleotides: The data from screening a set ofJNK1-specific phosphorothioate oligonucleotides (Table 2) indicate thefollowing results. Oligonucleotides showing activity in this assay, asreflected by levels of inhibition of JNK1 mRNA levels of at least 50%,include ISIS Nos. 11982, 11983, 11985, 11987, 12463, 12464, 12538,12539, 12548, 12549, 12550, 12552, 12553, 12554, 12555, 12556 and 12557(SEQ ID NOS: 5, 6, 8, 10, 13, 14, 15, 16, 17, 18, 19, 21, 22, 23, 24, 25and 26, respectively). These oligonucleotides are thus preferredembodiments of the invention for modulating JNK1 expression.Oligonucleotides showing levels of inhibition of JNK1 mRNAs of at least80% in this assay, include ISIS Nos. 11982, 12539, 12464, 12548, 12554and 12464 (SEQ ID NOS: 5, 14, 16, 17 and 23, respectively). Theseoligonucleotides are thus more preferred embodiments of the inventionfor modulating JNK1 expression.

The time course of inhibition of JNK1 mRNA expression by ISIS 12539 (SEQID NO: 16) is shown in Table 3. Following the 4 hour treatment with ISIS12539, the level of inhibition of JNK1 was greater than about 85% (t=0h), rose to about 95% inhibition at t=4 h, and subsequently remained atgreater than or equal to about 80% (t=12 and 48 h) or 60% (t=72 h).

TABLE 2 Activities of JNK1 Oligonucleotides GENE SEQ ID TARGET ISISNo:NO: REGION % EXPRESSION: % INHIBITION: 11978 1 ORF 85% 15% 11979 2 ORF90% 10% 11980 3 ORF 85% 15% 11981 4 ORF 62% 28% 11982 5 3′-UTR 13% 87%11983 6 3′-UTR 40% 60% 11984 7 3′-UTR 53% 47% 11985 8 3′-UTR 47% 53%11986 9 3′-UTR 90% 10% 11987 10 3′-UTR 47% 53% 11988 11 3′-UTR 78% 22%11989 12 3′-UTR 60% 40% 12463 13 tIR 23% 77% 12464 14 ORF 18% 82% 1253815 ORF 33% 67% 12539 16 ORF 9% 91% 12548 17 ORF 5% 95% 12549 18 ORF 28%72% 12550 19 ORF 40% 60% 12551 20 ORF 52% 48% 12552 21 ORF 34% 66% 1255322 ORF 25% 75% 12554 23 ORF 11% 89% 12555 24 ORF 27% 73% 12556 25 3′-UTR41% 59% 12557 26 3′-UTR 29% 71%

TABLE 3 Time Course of Response to JNK1 Antisense Oligonucleotides(ASOs) SEQ Normalized ID % ISIS # NO: ASO Description Time Control %Inhibition control — (LIPOFECTIN ™  0 h 100.0 0.0 only) control —(LIPOFECTIN ™  4 h 100.0 0.0 only) control — (LIPOFECTIN ™ 12 h 100.00.0 only) control — (LIPOFECTIN ™ 48 h 100.0 0.0 only) control —(LIPOFECTIN ™ 72 h 100.0 0.0 only) 12539 16 JNK1 active  0 h 14.1 85.912539 16 ″  4 h 5.9 94.1 12539 16 ″ 12 h 11.6 88.4 12539 16 ″ 48 h 21.079.0 12539 16 ″ 272 h  41.5 58.5

Additional JNK1 oligonucleotides: The results for JNK1-specificoligonucleotides (Table 2) indicate that one of the most activephosphorothioate oligonucleotides for modulating JNK1 expression is ISIS12539 (SEQ ID NO: 16). As detailed in Table 4, additionaloligonucleotides based on this oligonucleotide were designed to confirmand extend the findings described above.

Oligonucleotides ISIS Nos. 14320 (SEQ ID NO: 27) and 14321 (SEQ ID NO:28) are 2′-deoxy-phosphorothioate sense strand and scrambled controlsfor ISIS 12539 (SEQ ID NO: 16), respectively. ISIS Nos. 15346 and 15347are “gapmers” corresponding to ISIS 12539; both have 2′-methoxyethoxy“wings” (having phosphorothioate linkages in the case of ISIS 15346 andphosphodiester linkages in the case of ISIS 15347) and a central2′-deoxy “gap” designed to support RNaseH activity on the target mRNAmolecule. Similarly, ISIS Nos. 15348 to 15350 are “wingmers”corresponding to ISIS 12539 and have a 5′ or 3′ 2′-methoxyethoxyRNaseH-refractory “wing” and a 3′ or 5′ (respectively) 2′-deoxy “wing”designed to support RNaseH activity on the target JNK1 mRNA.

The chemically modified derivatives of ISIS 12539 (SEQ ID NO: 16) weretested in the Northern assay described herein at concentrations of 100and 400 nM, and the data (Table 5) indicate the following results. At400 nM, relative to the 2′-unmodified oligonucleotide ISIS 12539, both“gapmers” (ISIS Nos. 15346 and 15347) effected inhibition of JNK1 mRNAexpression up to at least about 88% inhibition. Similarly, the four“wingmers” (ISIS Nos. 15348 to 15351) effected inhibition of JNK1expression of up to at least about 60 to 70% inhibition.

TABLE 4 Chemically Modified JNK1 Oligonucleotides ISISNUCLEOTIDE SEQUENCE (5′ -> 3′)AND SEQID NO. CHEMICAL MODIFICATIONS* NO:COMMENTS 12539C^(S)T^(S)C^(S)T^(S)C^(S)T^(S)G^(S)T^(S)A^(S)G^(S)G^(S)C^(S)C^(S)C^(S)G^(S)C^(S)T^(S)T^(S)G^(S)G16 active 14320C^(S)C^(S)A^(S)A^(S)G^(S)C^(S)G^(S)G^(S)G^(S)C^(S)C^(S)T^(S)A^(S)C^(S)A^(S)G^(S)A^(S)G^(S)A^(S)G27 12539 sense control 14321C^(S)T^(S)T^(S)T^(S)C^(S)C^(S)G^(S)T^(S)T^(S)G^(S)G^(S)A^(S)C^(S)C^(S)C^(S)C^(S)T^(S)G^(S)G^(S)G28 scrambled control 15345 C ^(S) T ^(S) C ^(S) T ^(S) C ^(S) T ^(S) G^(S) T ^(S) A ^(S) G ^(S) G ^(S) C ^(S) C ^(S) C ^(S) G ^(S) C ^(S) T^(S) T ^(S) G ^(S) G 16 fully 2′- methoxyethoxy 15346 C ^(S) T ^(S) C^(S) T^(S)C^(S)T^(S)G^(S)T^(S)A^(S)G^(S)G^(S)C^(S)C^(S)C^(S)G^(S)C^(S)T^(S)T^(S)G^(S)G16 “gapmer” 15347 C ^(O) T ^(O) C ^(O) T ^(O) C^(S)T^(S)G^(S)T^(S)A^(S)G^(S)G^(S)C^(S)C^(S)C^(S) G ^(O) C ^(O) T ^(O) T^(O) G ^(O) G 16 “gapmer” 15348 C ^(S) T ^(S) C ^(S) T ^(S) C ^(S) T^(S) G ^(S) T ^(S) A ^(S) G ^(S) G ^(S)C^(S)C^(S)C^(S) G ^(S) C ^(S) T^(S) T ^(S) G ^(S) G 16 “wingmer” 15349C^(S)T^(S)C^(S)T^(S)C^(S)T^(S)G^(S)T^(S)A^(S) G ^(S) G ^(S) C ^(S) C^(S) C ^(S) G ^(S) C ^(S) T ^(S) T ^(S) G ^(S) G 16 “wingmer” 15351 C^(O) T ^(O) C ^(O) T ^(O) C ^(O) T ^(O) G ^(O) T ^(O) A ^(O) G ^(O) G^(S)C^(S)C^(S)C^(S)G^(S)C^(S)T^(S)T^(S)G^(S)G 16 “wingmer” 15350C^(S)T^(S)C^(S)T^(S)C^(S)T^(S)G^(S)T^(S)A^(S) G ^(O) G ^(O) C ^(O) C^(O) C ^(O) G ^(O) C ^(O) T ^(O) T ^(O) G ^(O) G 16 “wingmer” 20571 C^(S) T ^(S) C ^(S) T ^(S)C^(S)T^(S)G^(S)T^(S)A^(S)G^(S)G^(S) C ^(S) C^(S) C ^(S) G ^(S) C ^(S) T ^(S) T ^(S) G ^(S) G 1 fully 5-methyl-cytosine version of ISIS 15346 *Emboldened residues,2′-methoxyethoxy-residues (others are 2′-deoxy-) including “C” residues,5-methyl-cytosines; “^(O)”, phosphodiester linkage; “^(S)”,phosphorothioate linkage. --- “C” residues, 2′-deoxy 5-methylcytosineresidues;

TABLE 5 Activity of Chemically Modified JNK1 Antisense OligonucleotidesSEQ Normalized ID % ISIS # NO: Oligonucleotide Description* Dose Controlcontrol — No oligonucleotide — 100.0 (LIPOFECTIN ™ only) 12539 16 JNK1active, fully P═S & 100 nM 56.4 12539 16 fully 2′-deoxy 400 nM 26.715345 16 fully P═S & fully 2′-MOE 100 nM 95.4 15345 16 400 nM 89.1 1534616 gapmer: P═S, 2′-MOE wings; 100 nM 22.6 15346 16 P═S, 2′-deoxy core400 nM 11.0 15347 16 gapmer: P═O, 2′-MOE wings; 100 nM 27.1 15347 16P═S, 2-deoxy core 400 nM 11.7 15348 16 wingmer: fully P═S; 100 nM 30.415348 16 5′ 2′-MOE; 3′ 2-deoxy 400 nM 32.9 15349 16 wingmer: fully P═S;100 nM 42.5 15349 16 5′ 2-deoxy; 3′ 2′-MOE 400 nM 35.5 15351 16 wingmer:5′ P═O & 2′-MOE; 100 nM 45.1 15351 16 3′ P═S & 2-deoxy 400 nM 39.8 1535016 wingmer: 5′ P═S & 2′- 100 nM 71.1 15350 16 deoxy; 3′ P═O & 2′-MOE 400nM 41.3 *Abbreviations: P═O, phosphodiester linkage; P═S,hosphorothioate linkage; MOE, methoxyethoxy-.

Dose- and sequence-dependent response to JNK1 oligonucleotides: In orderto demonstrate a dose-dependent response to ISIS 12539 (SEQ ID NO: 16),different concentrations (i.e., 50, 100, 200 and 400 nM) of ISIS 12539were tested for their effect on JNK1 mRNA levels in A549 cells (Table6). In addition, two control oligonucleotides (ISIS 14320, SEQ ID NO:27, sense control, and ISIS 14321, SEQ ID NO: 28, scrambled control; seealso Table 4) were also applied to A549 cells in order to demonstratethe specificity of ISIS 12539. The results (Table 6) demonstrate thatthe response of A549 cells to ISIS 12539 is dependent on dose in anapproximately linear fashion. In contrast, neither of the controloligonucleotides effect any consistent response on JNK1 mRNA levels.

Western Assays: In order to assess the effect of oligonucleotidestargeted to JNK1 mRNAs on JNK1 protein levels, Western assays wereperformed essentially as described above in Example 2, with thefollowing exception(s) and/or modification(s). A primary antibody thatspecifically binds to JNK1 (catalog No. sc-474-G) was purchased fromSanta Cruz Biotechnology, Inc. (Santa Cruz, Calif.; other JNK1-specificantibodies are available from StressGen Biotechnologies, Inc., Victoria,BC, Canada; and Research Diagnostics, Inc., Flanders, N.J.). In thisexperiment, cells were grown and treated with oligonucleotide at 300 nMfor the initial 20 hours and then at 200 nM for 4 hours. At t=48 h,aliquots were removed for Northern and Western analyses, and fresh mediawas added to the cells. Aliquots for analysis were also taken at t=72 h.The samples from t=48 h and t=72 h were analyzed using the Northern andWestern assays described above.

TABLE 6 Dose-Dependent Responses to JNK1 Antisense Oligonucleotides SEQID Normalized % ISIS # NO: Oligonucleotide Description Dose Controlcontrol — No oligonucleotide — 100.0 (LIPOFECTIN ™ only) 12539 16 JNK1active  50 nM 70.3 12539 16 ″ 100 nM 51.6 12539 16 ″ 200 nM 22.4 1253916 ″ 400 nM 11.1 14320 27 12539 sense control  50 nM 103.6 14320 27 ″100 nM 76.3 14320 27 ″ 200 nM 98.9 14320 27 ″ 400 nM 97.1 14321 28 12539scrambled control  50 nM 91.8 14321 28 ″ 100 nM 94.1 14321 28 ″ 200 nM100.2 14321 28 ″ 400 nM 79.2

The data (Table 7) indicate the following results. In this assay, att=48 h, oligonucleotides showing a level of mRNA % inhibitionfrom >about 70% to about 100% include ISIS Nos. 12539 (phosphorothioatelinkages), 15346 and 15347 (“gapmers”), and 15348 and 15351 (5′“wingmers”) (SEQ ID NO: 16). Oligonucleotides showing levels of mRNAinhibition of from ≧about 90% to about 100% of JNK1 mRNAs in this assayinclude ISIS Nos. 12539, 15345 AND 15346 (SEQ ID NO: 16). Theoligonucleotides tested showed approximately parallel levels of JNK1protein inhibition; ISIS Nos. 12539, 15346-15348 and 15351 effectedlevels of protein inhibition ≧about 40%, and ISIS Nos. 12539, 15346 and15347 effected levels of protein inhibition ≧about 55%.

At t=72 h, oligonucleotides showing a level of mRNA % inhibitionfrom >about 70% to about 100% include ISIS Nos. 12539 (phosphorothioatelinkages), 15346 and 15347 (“gapmers”), and 15348 (5′ “wingmers”) (SEQID NO: 16). Oligonucleotides showing levels of mRNA inhibition of from≧about 90% to about 100% of JNK1 mRNAs at this point in the assayinclude ISIS Nos. 12539 and 15346 (SEQ ID NO: 16). Overall, theoligonucleotides tested showed higher levels of JNK1 protein inhibitionat this point in the assay. With the exception of the fully2′-methoxyethoxy-modified ISIS 15345, all of the oligonucleotides inTable 7 effect ≧about 40% protein inhibition. ISIS Nos. 12539,15346-15348 and 15351 effected levels of protein inhibition ≧about 60%,and ISIS Nos. 12539, 15346 and 15347 effected levels of proteininhibition ≧about 70%.

TABLE 7 Modulation of JNK1 mRNA and JNK1 Protein Levels by Modified JNK1Antisense Oligonucleotides SEQ Protein ID RNA % Protein ISIS # NO: RNA %Control % Inhibition Control % Inhibition t = 48 h control — 100.0 0.0100.0 0.0 12539 16 6.7 93.3 44.3 55.7 15345 16 70.3 29.7 105.0 (0.0)15346 16 4.3 95.7 42.7 57.3 15347 16 7.9 92.1 38.8 61.2 15348 16 24.375.7 58.3 41.7 15349 16 63.1 36.9 69.5 30.5 15350 16 49.2 50.8 71.7 28.315351 16 26.9 73.1 52.4 47.6 t = 72 h control 16 100.0 0.0 100.0 0.012539 16 11.7 88.3 29.2 70.8 15345 16 187.4 (0.0) 87.8 12.2 15346 1610.6 89.4 25.7 74.3 15347 16 8.2 81.8 28.4 71.6 15348 16 28.0 72.0 41.758.3 15349 16 52.0 48.0 56.5 43.5 15350 16 54.4 45.6 58.4 41.6 15351 1646.1 53.9 37.0 63.0

Oligonucleotides specific for JNK1 isoforms: Subsequent to the initialdescriptions of JNK1 (Derijard et al., Cell, 1994, 76, 1025), cDNAsencoding related isoforms of JNK1 were cloned and their nucleotidesequences determined (Gupta et al., EMBO Journal, 1996, 15, 2760). Inaddition to JNK1-a1 (GenBank accession No. L26318 (SEQ ID NO: 87), locusname “HUMJNK1”), which encodes a polypeptide having an amino acidsequence identical to that of JNK1, the additional isoforms includeJNK1-a2 (GenBank accession No. U34822 (SEQ ID NO: 89), locus name“U34822”), JNK1-β1 (GenBank accession No. U35004 (SEQ ID NO: 90), locusname “HSU35004”) and JNK1-β2 (GenBank accession No. U35005 (SEQ ID NO:91), locus name “HSU35005”). The four isoforms of JNK1, which probablyarise from alternative mRNA splicing, can each interact with differenttranscription factors or sets of transcription factors (Gupta et al.,EMBO Journal, 1996, 15, 2760). As detailed below, the oligonucleotidesof the invention are specific for certain members or sets of theseisoforms of JNK1.

In the ORFs of mRNAs encoding JNK1/JNK1-a1 and JNK1-a2, nucleotides (nt)631-665 of JNK1/JNK1-a1 (Genbank accession No. L26318 (SEQ ID NO: 87))and nt 625-659 of JNK1-a2 (Genbank accession No. U34822 (SEQ ID NO: 89))have the sequence shown below as SEQ ID NO: 63, whereas, in the ORFs ofmRNAs encoding JNK1-β1 and JNK1-β2, nt 631-665 of JNK1-β1 (GenBankaccession No. U35004 (SEQ ID NO: 90)) and nt 626-660 of JNK1-β2 (GenBankaccession No. U35005 (SEQ ID NO: 91)) have the sequence shown below asSEQ ID NO: 64. For purposes of illustration, SEQ ID NOS: 63 and 64 areshown aligned with each other (vertical marks, “|,” indicate bases thatare identical in both sequences):

Due to this divergence between the a and b JNK1 isoforms, antisenseoligonucleotides derived from the reverse complement of SEQ ID NO: 63(i.e., SEQ ID NO: 65, see below) can be used to modulate the expressionof JNK1/JNK1-a1 and JNK1-a2 without significantly effecting theexpression of JNK1-β1 and JNK1-β2. In like fashion, antisenseoligonucleotides derived from the reverse complement of SEQ ID NO: 64(i.e., SEQ ID NO: 66, see below) can be selected and used to modulatethe expression of JNK1-β1 and JNK1-β2 without significantly effectingthe expression of JNK1/JNK1-a1 and JNK1-a2. As an example, anoligonucleotide having a sequence derived from SEQ ID NO: 65 but not toSEQ ID NO: 66 is specifically hybridizable to mRNAs encodingJNK1/JNK1-a1 and JNK1-a2 but not to those encoding JNK1-β1 and JNK1-β2:

As a further example, in the ORFs of mRNAs encoding JNK1/JNK1-a1 andJNK1-a2, nt 668-711 of JNK1/JNK1-a1 (Genbank accession No. L26318 (SEQID NO: 87)) and nt 662-705 of JNK1-a2 (Genbank accession No. U34822 (SEQID NO: 89)) have the sequence shown below as SEQ ID NO: 67, whereas, inthe ORFs of mRNAs encoding JNK1-β1 and JNK1-β2, nt 668-711 of JNK1-β1(GenBank accession No. U35004 (SEQ ID NO: 90)) and nt 663-706 of JNK1-β2(GenBank accession No. U35005 (SEQ ID NO: 91)) have the sequence shownbelow as SEQ ID NO: 68. For purposes of illustration, SEQ ID NOS: 67 and68 are shown aligned with each other as follows:

Due to this divergence between the a and b JNK1 isoforms, antisenseoligonucleotides derived from the reverse complement of SEQ ID NO: 67(i.e., SEQ ID NO: 69, see below) are specifically hybridizable to mRNAsencoding, and can be selected and used to modulate the expression of,JNK1/JNK1-a1 and JNK1-a2 without significantly effecting the expressionof JNK1-β1 and JNK1-β2. In like fashion, antisense oligonucleotidesderived from the reverse complement of SEQ ID NO: 68 (i.e., SEQ ID NO:70, see below) are specifically hybridizable to mRNAs encoding, and canbe selected and used to modulate the expression of, can be selected andused to modulate the expression of JNK1-β1 and JNK1-β2 withoutsignificantly effecting the expression of JNK/JNK1-a1 and JNK1-a2:

In the case of the carboxyl terminal portion of the JNK1 isoforms,JNK1/JNK1-a1 shares identity with JNK1-β1; similarly, JNK1-a2 andJNK1-β2 have identical carboxy terminal portions. The substantialdifferences in the amino acid sequences of these isoforms (5 amino acidsin JNK1/JNK1-a1 and JNK1-β1 are replaced with 48 amino acids in JNK1-a2and JNK1-β2) result from a slight difference in nucleotide sequence thatshifts the reading frame. Specifically, in the ORFs of mRNAs encodingJNK1/JNK1-a1 and JNK1-β1, nt 1144-1175 of JNK1/JNK1-a1 (Genbankaccession No. L26318 (SEQ ID NO: 87)) and JNK1-β1 (Genbank accession No.U35004 (SEQ ID NO: 90)) have the sequence shown below as SEQ ID NO: 71,whereas, in the ORFs of mRNAs encoding JNK1-a2 and JNK1-β2, nt 1138-1164of JNK1-a2 (GenBank accession No. U34822 (SEQ ID NO: 89)) and nt1139-1165 of JNK1-β2 (GenBank accession No. U35005 (SEQ ID NO: 91)) havethe sequence shown below as SEQ ID NO: 72. For purposes of illustration,SEQ ID NOS: 71 and 72 are shown aligned with each other (dashes, A-,”indicate bases that are absent in the indicated sequence, and emboldenedbases indicate the stop codon for the JNK1/JNK1-a1 and JNK1-β1 ORFs):

Due to this divergence between the JNK1 isoforms, antisenseoligonucleotides derived from the reverse complement of SEQ ID NO: 71(i.e., SEQ ID NO: 73, see below) are specifically hybridizable to mRNAsencoding, and can be selected and used to modulate the expression of,JNK1/JNK1-a1 and JNK1-β1 without significantly effecting the expressionof JNK1-a2 and JNK1-β2. In like fashion, antisense oligonucleotidesderived from the reverse complement of SEQ ID NO: 72 (i.e., SEQ ID NO:74, see below) are specifically hybridizable to mRNAs encoding, and canbe selected and used to modulate the expression of, JNK1-a2 and JNK1-β2without significantly effecting the expression of JNK1/JNK1-a1 andJNK1-β1:

In preferred embodiments, such isoform-specific

oligonucleotides such as are described above are methoxyethoxy “gapmers”or “wingmers” in which the RNase H-sensitive “gap” or “wing” ispositioned so as to overlap a region of nonidentity in the aboveantisense sequences, i.e., SEQ ID NOS: 65, 66, 69, 70, 73 and 74.

Example 4 Oligonucleotide-Mediated Inhibition of JNK2 Expression

JNK2 oligonucleotide sequences: Table 8 lists the nucleotide sequencesof oligonucleotides designed to specifically hybridize to JNK2 mRNAs andthe corresponding ISIS and SEQ ID numbers thereof. The target genenucleotide co-ordinates and gene target region are also included. Thenucleotide co-ordinates are derived from GenBank accession No. L31951(SEQ ID NO: 92), locus name “HUMJNK2” (see also FIG. 1(A) of Sluss etal., Mol. Cel. Biol., 1994, 14, 8376, and Kallunki et al., Genes &Development, 1994, 8, 2996). The abbreviations for gene target regionsare as follows: 5′-UTR, 5′ untranslated region; tIR, translationinitiation region; ORF, open reading frame; 3′-UTR, 3′ untranslatedregion. The nucleotides of the oligonucleotides whose sequences arepresented in Table 8 are connected by phosphorothioate linkages and areunmodified at the 2′ position (i.e., 2-deoxy). It should be noted thatthe oligonucleotide target co-ordinate positions and gene target regionscan vary within mRNAs encoding related isoforms of JNK2 (see subsectionG, below).

In addition to hybridizing to human JNK2 mRNAs, the full oligonucleotidesequence of ISIS No. 12562 (SEQ ID NO: 33) hybridizes to the ORF ofmRNAs from Rattus norvegicus that encode a stress-activated proteinkinase named “p54a2” (Kyriakis et al., Nature, 1994, 369, 156).Specifically, ISIS 12562 (SEQ ID NO: 33) hybridizes to bases 649-668 ofGenBank accession No. L27112 (SEQ ID NO: 93), locus name “RATSAPKB.”This oligonucleotide is thus a preferred embodiment of the invention forinvestigating the role of the p54a2 protein kinase in rat in vitro,i.e., in cultured cells or tissues derived from whole animals, or invivo.

JNK2-specific probes: In initial screenings of a set of oligonucleotidesderived from the JNK2 sequence (Table 9) for biological activity, a cDNAclone of JNK2 (Kallunki et al., Genes & Development, 1994, 8, 2996) wasradiolabeled and used as a JNK2-specific probe in Northern blots.Alternatively, however, one or more of the oligonucleotides of Table 8is detectably labeled and used as a JNK2-specific probe.

Activities of JNK2 oligonucleotides: The data from screening a set ofJNK2-specific phosphorothioate oligonucleotides (Table 9) indicate thefollowing results. Oligonucleotides showing activity in this assay, asreflected by levels of inhibition of JNK2 mRNA levels of at least 50%,include ISIS Nos. 12558, 12559, 12560, 12563, 12564, 12565, 12566,12567, 12568, 12569 and 12570 (SEQ ID NOS: 29, 30, 31, 34, 35, 36, 37,38, 39, 40 and 41, respectively). These oligonucleotides are thuspreferred embodiments of the invention for modulating JNK2 expression.Oligonucleotides showing levels of JNK2 mRNAs of at least 80% in thisassay, include ISIS Nos. 12558, 12560, 12565, 12567, 12568 and 12569(SEQ ID NOS: 29, 31, 36, 38, 39 and 40, respectively). Theseoligonucleotides are thus more preferred embodiments of the inventionfor modulating JNK2 expression.

The time course of inhibition of JNK2 mRNA expression by ISIS 12560 (SEQID NO: 31) is shown in Table 10. Following the 4 hour treatment withISIS 12560, the level of inhibition of JNK2 was greater than or equal toabout 80% for at least about 12 hours and greater than or equal to about60% up to at least about t=48 h.

TABLE 8 Nucleotide Sequences of JNK2 Oligonucleotides TARGET GENENUCLEO-SEQID TIDECO- GENETARGET ISISNO. NUCLEOTIDE SEQUENCE(5′ -> 3′) NO:ORDINATES REGION 12558 GTT-TCA-GAT-CCC-TCG-CCC-GC 29 0003-0022 5′-UTR12559 TGC-AGC-ACA-AAC-AAT-CCC-TT 30 0168-0187 ORF 12560GTC-CGG-GCC-AGG-CCA-AAG-TC 31 0563-0582 ORF 12561CAG-GAT-GAC-TTC-GGG-CGC-CC 32 0633-0652 ORF 12562GCT-CTC-CCA-TGA-TGC-AAC-CC 33 0691-0710 ORF 12563ATG-GGT-GAC-GCA-GAG-CTT-CG 34 0997-1016 ORF 12564CTG-CTG-CAT-CTG-AAG-GCT-GA 35 1180-1199 ORF 12565TGA-GAA-GGA-GTG-GCG-TTG-CT 36 1205-1224 ORF 12566TGC-TGT-CTG-TGT-CTG-AGG-CC 37 1273-1292 ORF 12567GGT-CCC-GTC-GAG-GCA-TCA-AG 38 1295-1314 ORF 12568CAT-TTC-AGG-CCC-ACG-GAG-GT 39 1376-1395 3′-UTR 12569GGT-CTG-AAT-AGG-GCA-AGG-CA 40 1547-1566 3′-UTR 12570GGG-CAA-GTC-CAA-GCA-AGC-AT 41 1669-1688 3′-UTR

TABLE 9 Activities of JNK2 Oligonucleotides GENE SEQ ID TARGET ISIS NO.NO: REGION % EXPRESSION % INHIBITION 12558 29 5′-UTR 15% 85% 12559 30ORF 28% 72% 12560 31 ORF 11% 89% 12561 32 ORF 60% 40% 12562 33 ORF 89%11% 12563 34 ORF 22% 78% 12564 35 ORF 28% 72% 12565 36 ORF 19% 81% 1256637 ORF 42% 58% 12567 38 ORF 18% 82% 12568 39 3′-UTR 20% 80% 12569 403′-UTR 13% 87% 12570 41 3′-UTR 24% 76%

TABLE 10 Time Course of Response to JNK2 Antisense Oligonucleotides(ASOs) Normalized SEQ ID ASO % % ISIS # NO: Description Time ControlInhibition control — (LIPOFECTIN ™  0 h 100.0 0.0 only) control —(LIPOFECTIN ™  4 h 100.0 0.0 only) control — (LIPOFECTIN ™ 12 h 100.00.0 only) control — (LIPOFECTIN ™ 48 h 100.0 0.0 only) control —(LIPOFECTIN ™ 72 h 100.0 0.0 only) 12560 31 JNK2 active  0 h 20.2 79.812560 31 ″  4 h 11.1 88.9 12560 31 ″ 12 h 21.8 78.2 12560 31 ″ 48 h 42.757.3 12560 31 ″ 72 h 116.8 (0.0)

Additional JNK2 oligonucleotides: The results for JNK2-specificoligonucleotides (Table 9) indicate that one of the most activephosphorothioate oligonucleotides for modulating JNK2 expression is ISIS12560 (SEQ ID NO: 31). As detailed in Table 11, additionaloligonucleotides based on this oligonucleotide were designed to confirmand extend the findings described above.

Oligonucleotides ISIS Nos. 14318 (SEQ ID NO: 42) and 14319 (SEQ ID NO:43) are 2′-deoxy-phosphorothioate sense strand and scrambled controlsfor ISIS 12560 (SEQ ID NO: 31), respectively. ISIS Nos. 15353 and 15354are “gapmers” corresponding to ISIS 12560; both have 2′-methoxyethoxy“wings” (having phosphorothioate linkages in the case of ISIS 15353 andphosphodiester linkages in the case of ISIS 15354) and a central2′-deoxy “gap” designed to support RNaseH activity on the target mRNAmolecule. Similarly, ISIS Nos. 15355 to 15358 are “wingmers”corresponding to ISIS 12560 and have a 5′ or 3′ 2′-methoxyethoxyRNaseH-refractory “wing” and a 3′ or 5′ (respectively) 2-deoxy “wing”designed to support RNaseH activity on the target JNK2 mRNA.

The chemically modified derivatives of ISIS 12560 (SEQ ID NO: 31) weretested in the Northern assay described herein at concentrations of 100and 400 nM, and the data (Table 12) indicate the following results. At400 nM, relative to the 2′-unmodified oligonucleotide ISIS 12560, both“gapmers” (ISIS Nos. 15353 and 15354) effected approximately 80%inhibition of JNK2 mRNA expression. Similarly, the four “wingmers” (ISISNos. 15355 to 15358) effected 70-90% inhibition of JNK2 expression.

Dose- and sequence-dependent response to JNK2 oligonucleotides: In orderto demonstrate a dose-dependent response to ISIS 12560 (SEQ ID NO: 31),different concentrations (i.e., 50, 100, 200 and 400 nM) of ISIS 12560were tested for their effect on JNK2 mRNA levels in A549 cells (Table13). In addition, two control oligonucleotides (ISIS 14318, SEQ ID NO:42, sense control, and ISIS 14319, SEQ ID NO: 43, scrambled control; seealso Table 11) were also applied to A549 cells in order to demonstratethe specificity of ISIS 12560. The results (Table 12) demonstrate thatthe response of A549 cells to ISIS 12539 is dependent on dose in anapproximately linear fashion. In contrast, neither of the controloligonucleotides effect any consistent response on JNK2 mRNA levels.

TABLE 11 Chemically Modified JNK2 OligonucleotidesNUCLEOTIDE SEQUENCE (5′ -> 3′)AND SEQ ID ISISNO. CHEMICAL MODIFICATIONS*NO: COMMENTS 12560G^(S)T^(S)C^(S)C^(S)G^(S)G^(S)G^(S)C^(S)C^(S)A^(S)G^(S)G^(S)C^(S)C^(S)A^(S)A^(S)A^(S)G^(S)T^(S)C31 active 14318G^(S)A^(S)C^(S)T^(S)T^(S)T^(S)G^(S)G^(S)C^(S)C^(S)T^(S)G^(S)G^(S)C^(S)C^(S)C^(S)G^(S)G^(S)A^(S)C42 12560 sense control 14319G^(S)T^(S)G^(S)C^(S)G^(S)C^(S)G^(S)C^(S)G^(S)A^(S)G^(S)C^(S)C^(S)C^(S)G^(S)A^(S)A^(S)A^(S)T^(S)C43 scrambled control 15352 G ^(S) T ^(S) C ^(S) C ^(S) G ^(S) G ^(S) G^(S) C ^(S) C ^(S) A ^(S) G ^(S) G ^(S) C ^(S) C ^(S) A ^(S) A ^(S) A^(S) G ^(S) T ^(S) C 31 fully 2′- methoxyethoxy 15353 G ^(S) T ^(S) C^(S) C ^(S) G ^(S)G^(S)G^(S)C^(S)C^(S)A^(S)G^(S)G^(S)C^(S)C^(S) A ^(S) A^(S) A ^(S) G ^(S) T ^(S) C 31 “gapmer” 15354 G ^(O) T ^(O) C ^(O) C^(O) G ^(S)G^(S)G^(S)C^(S)C^(S)A^(S)G^(S)G^(S)C^(S)C^(S) A ^(O) A ^(O) A^(O) G ^(O) T ^(O) C 31 “gapmer” 15355 G ^(S) T ^(S) C ^(S) C ^(S) G^(S) G ^(S) G ^(S) C ^(S) C ^(S) A ^(S) G^(S)G^(S)C^(S)C^(S)A^(S)A^(S)A^(S)G^(S)T^(S)C 31 “wingmer” 15356G^(S)T^(S)C^(S)C^(S)G^(S)G^(S)G^(S)C^(S)C^(S) A ^(S) G ^(S) G ^(S) C^(S) C ^(S) A ^(S) A ^(S) A ^(S) G ^(S) T ^(S) C 31 “wingmer” 15358 G^(O) T ^(O) C ^(O) C ^(O) G ^(O) G ^(O) G ^(O) C ^(O) C ^(O) A ^(O) G^(S)G^(S)C^(S)C^(S)A^(S)A^(S)A^(S)G^(S)T^(S)C 31 “wingmer” 15357G^(S)T^(S)C^(S)C^(S)G^(S)G^(S)G^(S)C^(S)C^(S) A ^(O) G ^(O) G ^(O) C^(O) C ^(O) A ^(O) A ^(O) A ^(O) G ^(O) T ^(O) C 31 “wingmer” 20572 G^(S) T ^(S) C ^(S) C ^(S) G ^(S)G^(S)G^(S) C ^(S) C ^(S)A^(S)G^(S)G^(S)C ^(S) C ^(S) A ^(S) A ^(S) A ^(S) G ^(S) T ^(S) C 31 fully 5-methyl-cytosine version of ISIS 15353 *Emboldened residues,2′-methoxyethoxy-residues (others are 2′-deoxy-) including “C” residues,5-methyl-cytosines; “^(O)”, phosphodiester linkage; “^(S)”,phosphorothioate linkage. --- “C” residues, 2′-deoxy 5-methylcytosineresidues; ---

TABLE 12 Activity of Chemically Modified JNK2 Antisense OligonucleotidesSEQ Normalized ID % ISIS # NO: Oligonucleotide Description Dose Controlcontrol — No oligonucleotide — 100.0 (LIPOFECTIN ™ only) 12560 31 JNK2active, fully P═S & 100 nM 62.1 12560 31 fully 2-deoxy 400 nM 31.4 1535231 fully P═S & fully 2′-MOE 100 nM 132.4 15352 31 400 nM 158.4 15353 31gapmer: P═S, 2′-MOE wings; 100 nM 56.7 15353 31 P═S, 2-deoxy core 400 nM21.2 15354 31 gapmer: P═O, 2′-MOE wings; 100 nM 38.3 15354 31 P═S,2-deoxy core 400 nM 17.1 15355 31 wingmer: fully P═S; 100 nM 61.3 1535531 5′ 2′-MOE; 3′ 2-deoxy 400 nM 29.1 15356 31 wingmer: fully P═S; 100 nM38.6 15356 31 5′ 2-deoxy; 3′ 2′-MOE 400 nM 11.0 15358 31 wingmer: 5′ P═O& 2′-MOE; 100 nM 47.4 15358 31 3′ P═S & 2-deoxy 400 nM 29.4 15357 31wingmer: 5′ P═S & 2′- 100 nM 42.8 15357 31 deoxy; 3′ P═O & 2′-MOE 400 nM13.7

TABLE 13 Dose-Dependent Responses to JNK2 Antisense Oligonucleotides SEQID Normalized % ISIS # NO: Oligonucleotide Description Dose Controlcontrol — No oligonucleotide — 100.0 (LIPOFECTIN ™ only) 12560 31 JNK2active  50 nM 68.1 12560 31 ″ 100 nM 50.0 12560 31 ″ 200 nM 25.1 1256031 ″ 400 nM 14.2 14318 42 12560 sense control  50 nM 87.1 14318 42 ″ 100nM 89.8 14318 42 ″ 200 nM 92.1 14318 42 ″ 400 nM 99.6 14319 43 12560scrambled control  50 nM 90.4 14319 43 ″ 100 nM 93.7 14319 43 ″ 200 nM110.2 14319 43 ″ 400 nM 100.0

Western Assays: In order to assess the effect of oligonucleotidestargeted to JNK2 mRNAs on JNK2 protein levels, Western assays areperformed essentially as described above in Examples 2 and 3. A primaryantibody that specifically binds to JNK2 is purchased from, for example,Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.; UpstateBiotechnology, Inc., Lake Placid, N.Y.; StressGen Biotechnologies, Inc.,Victoria, BC, Canada; or Research Diagnostics, Inc., Flanders, N.J.

Oligonucleotides specific for JNK2 isoforms: Subsequent to the initialdescriptions of JNK2 (Sluss et al., Mol. Cel. Biol., 1994, 14, 8376;Kallunki et al., Genes & Development, 1994, 8, 2996; GenBank accessionNo. HSU09759 (SEQ ID NO: 94), locus name “U09759 (SEQ ID NO: 94)”),cDNAs encoding related isoforms of JNK2 were cloned and their nucleotidesequences determined (Gupta et al., EMBO Journal, 1996, 15, 2760). Inaddition to JNK2-a2 (GenBank accession No. L31951 (SEQ ID NO: 92), locusname “HUMJNK2”), which encodes a polypeptide having an amino acidsequence identical to that of JNK2, the additional isoforms includeJNK2-a1 (GenBank accession No. U34821 (SEQ ID NO: 95), locus name“HSU34821”), JNK2-β1 (GenBank accession No. U35002 (SEQ ID NO: 96),locus name “HSU35002”) and JNK2-β2 (GenBank accession No. U35003 (SEQ IDNO: 97), locus name “HSU35003”). The four isoforms of JNK2, whichprobably arise from alternative mRNA splicing, can each interact withdifferent transcription factors or sets of transcription factors (Guptaet al., EMBO Journal, 1996, 15, 2760). As detailed below, theoligonucleotides of the invention are specific for certain members orsets of these isoforms of JNK2.

In the ORFs of mRNAs encoding JNK2/JNK2-a2 and JNK2-a1, nucleotides (nt)689-748 of JNK2/JNK2-a2 (GenBank accession No. L31951 (SEQ ID NO: 92))and nt 675-734 of JNK2-a1 (GenBank accession No. U34821 (SEQ ID NO: 95))have the sequence shown below as SEQ ID NO: 75, whereas, in the ORFs ofmRNAs encoding JNK2-β1 and JNK2-β2, nt 653-712 of JNK2-β1 (GenBankaccession No. U35002 (SEQ ID NO: 96)) and nt 665-724 of JNK2-β2 (GenBankaccession No. U35003 (SEQ ID NO: 97)) have the sequence shown below asSEQ ID NO: 76. For purposes of illustration, SEQ ID NOS: 75 and 76 areshown aligned with each other (vertical marks, “|,” indicate bases thatare identical in both sequences):

Due to this divergence between the a and b JNK2 isoforms, antisenseoligonucleotides derived from the reverse complement of SEQ ID NO: 75(i.e., SEQ ID NO: 77, see below) are specifically hybridizable to, andcan be selected and used to modulate the expression of, JNK2/JNK2-a2 andJNK2-a1 without significantly effecting the expression of JNK1-β1 andJNK1-β2. In like fashion, antisense oligonucleotides derived from thereverse complement of SEQ ID NO: 76 (i.e., SEQ ID NO: 78, see below) arespecifically hybridizable to, and can be selected and used to modulatethe expression of, JNK2-β1 and JNK2-β2 without significantly effectingthe expression of JNK2/JNK2-a2 and JNK2-a1. As an example, anoligonucleotide having a sequence derived from SEQ ID NO: 77 but notfrom SEQ ID NO: 78 is specifically hybridizable to, mRNAs encodingJNK1/JNK1-a1 and JNK1-a2 but not to those encoding JNK2-β1 and JNK2-β2:

In the case of the carboxyl terminal portion of the JNK2 isoforms,JNK2/JNK2-a2 shares identity with JNK1-β2; similarly, JNK2-a1 andJNK2-β1 have identical carboxy terminal portions. The substantialdifferences in the amino acid sequences of these isoforms (5 amino acidsin JNK2-a2 and JNK2-β2 are replaced with 47 amino acids in JNK2/JNK2-a2and JNK2-β2) result from a slight difference in nucleotide sequence thatshifts the reading frame. Specifically, in the ORFs of mRNAs encodingJNK2-a1 and JNK1-131, nt 1164-1198 of JNK2-a1 (GenBank accession No.U34821 (SEQ ID NO: 95)) and nt 1142-1176 of JNK2-β1 (GenBank accessionNo. U35002 (SEQ ID NO: 96)) have the sequence shown below as SEQ ID NO:79, whereas, in the ORFs of mRNAs encoding JNK2/JNK2-a2 and JNK2-β2, nt1178-1207 of JNK2/JNK2-a2 (GenBank accession No. L31951 (SEQ ID NO: 92))and nt 1154-1183 of JNK2-β2 (GenBank accession No. U35003 (SEQ ID NO:97)) have the sequence shown below as SEQ ID NO: 80. For purposes ofillustration, SEQ ID NOS: 79 and 80 are shown aligned with each other(dashes, “-,” indicate bases that are absent in the indicated sequence,and emboldened bases indicate the stop codon for the JNK2-a1 and JNK2-β1ORFs):

Due to this divergence between the JNK2 isoforms, antisenseoligonucleotides derived from the reverse complement of SEQ ID NO: 79(i.e., SEQ ID NO: 81, see below) are specifically hybridizable to, andcan be selected and used to modulate the expression of, mRNAs encodingJNK2-a1 and JNK2-β1 without significantly effecting the expression ofJNK2/JNK2-a2 and JNK2-β2. In like fashion, antisense oligonucleotidesderived from the reverse complement of SEQ ID NO: 80 (i.e., SEQ ID NO:82, see below) are specifically hybridizable to, and can be selected andused to modulate the expression of, mRNAs encoding JNK2/JNK2-a2 andJNK2-β2 without significantly effecting the expression of JNK2-a1 andJNK2-β1. As an example, ISIS 12564 (SEQ ID NO: 35) corresponds to SEQ IDNO: 82 but not to SEQ ID NO: 81, and is thus specifically hybridizableto, and can be used to modulate the expression of, mRNAs encodingJNK2/JNK2-a2 and JNK2-β2 but not those encoding JNK2-a1 and JNK2-a1:

In preferred embodiments, such isoform-specific oligonucleotides such asare described above are methoxyethoxy “gapmers” or “wingmers” in whichthe RNase H-sensitive “gap” or “wing” is positioned so as to overlap aregion of nonidentity in the above antisense sequences, i.e., SEQ IDNOS: 77, 78, 81 and 82.

Example 5 Oligonucleotide-Mediated Inhibition of JNK3 Expression

A. JNK3 oligonucleotide sequences: Table 14 lists the nucleotidesequences of oligonucleotides designed to specifically hybridize to JNK3mRNAs and the corresponding ISIS and SEQ ID numbers thereof. The targetgene nucleotide co-ordinates and gene target region are also included.The nucleotide co-ordinates are derived from GenBank accession No.U07620 (SEQ ID NO: 98), locus name “HSU07620” see also FIG. 4(A) ofMohit et al., Neuron, 1994, 14, 67). The abbreviations for gene targetregions are as follows: 5′-UTR, 5′ untranslated region; tIR, translationinitiation region; ORF, open reading frame; 3′-UTR, 3′ untranslatedregion. It should be noted that the oligonucleotide target co-ordinatepositions and gene target regions can vary within mRNAs encoding relatedisoforms of JNK3 (see subsection D, below).

The nucleotides of the oligonucleotides whose sequences are presented inTable 14 are connected by phosphorothioate linkages and are “gapmers.”Specifically, the six nucleotides of the 3′ and 5′ termini are2′-methoxyethoxy-modified and are shown emboldened in Table 14, whereasthe central eight nucleotides are unmodified at the 2′ position (i.e.,2-deoxy).

In addition to hybridizing to human JNK3 mRNAs, the full oligonucleotidesequences of ISIS Nos. 16692, 16693, 16703, 16704, 16705, 16707, and16708 (SEQ ID NOS: 46, 47, 56, 57, 58, 60 and 61, respectively)specifically hybridize to mRNAs from Rattus norvegicus that encode astress-activated protein kinase named “p54β” (Kyriakis et al., Nature,1994, 369, 156; GenBank accession No. L27128 (SEQ ID NO: 99), locus name“RATSAPKC.” Furthermore, the full oligonucleotide sequences of 16692,16693, 16695, 16703, 16704, 16705, 16707 and 16708 (SEQ ID NOS: 46, 47,49, 56, 57, 58, 60 and 61, respectively) specifically hybridize to mRNAsfrom Mus musculus that encode a mitogen activated protein (MAP) kinasestress activated protein named the “p459^(3F12) SAP kinase” (Martin etal., Brain Res. Mol. Brain. Res., 1996, 35, 47; GenBank accession No.L35236 (SEQ ID NO: 100), locus name “MUSMAPK”). These oligonucleotidesare thus preferred embodiments of the invention for investigating therole of the p54β and p459^(3F12) SAP protein kinases in rat or mouse,respectively, in vitro, i.e., in cultured cells or tissues derived fromwhole animals or in vivo. The target gene nucleotide co-ordinates andgene target regions for these oligonucleotides, as defined for theseGenBank entries, are detailed in Table 15.

JNK3-specific probes: In initial screenings of a set of oligonucleotidesderived from the JNK3 sequence for biological activity, a cDNA clone ofJNK3 (Derijard et al., Cell, 1994, 76, 1025) was radiolabeled and usedas a JNK3-specific probe in Northern blots. Alternatively, however, oneor more of the oligonucleotides of Table 14 is detectably labeled andused as a JNK3-specific probe.

Western Assays: In order to assess the effect of oligonucleotidestargeted to JNK3 mRNAs on JNK3 protein levels, Western assays areperformed essentially as described above in Examples 2 through 4. Aprimary antibody that specifically binds to JNK3 is purchased from, forexample, Upstate Biotechnology, Inc. (Lake Placid, N.Y.), StressGenBiotechnologies Corp. (Victoria, BC, Canada), or New England Biolabs,Inc. (Beverly, Mass.).

TABLE 14 Nucleotide Sequences of JNK3 Oligonucleotides TARGET GENENUCLEOTIDE SEQID CO- GENETARGET ISISNO. NUCLEOTIDE SEQUENCE¹(5′ -> 3′)NO: ORDINATES REGION 16690 TTC-AAC-AGT-TTC-TTG-CAT-AA 44 0157-01765′-UTR 16691 CTC-ATC-TAT-AGG-AAA-CGG-GT 45 0182-0200 5′-UTR 16692TGG-AGG-CTC-ATA-AAT-ACC-AC 46 0215-0234 tIR 16693TAT-AAG-AAA-TGG-AGG-CTC-AT 47 0224-0243 tIR 16694TCA-CAT-CCA-ATG-TTG-GTT-CA 48 0253-0272 ORF 16695TTA-TCG-AAT-CCC-TGA-CAA-AA 49 0281-0300 ORF 16696GTT-TGG-CAA-TAT-ATG-ACA-CA 50 0310-0329 ORF 16697CTG-TCA-AGG-ACA-GCA-TCA-TA 51 0467-0486 ORF 16698AAT-CAC-TTG-ACA-TAA-GTT-GG 52 0675-0694 ORF 16699TAA-ATC-CCT-GTG-AAT-AAT-TC 53 0774-0793 ORF 16700GCA-TCC-CAC-AGA-CCA-TAT-AT 54 0957-0976 ORF 16702TGT-TCT-CTT-TCA-TCC-AAC-TG 55 1358-1377 ORF 16703TCT-CAC-TGC-TGT-TCA-CTG-CT 56 1485-1504 tIR 16704GGG-TCT-GGT-CGG-TGG-ACA-TG 57 1542-1561 3′-UTR 16705AGG-CTG-CTG-TCA-GTG-TCA-GA 58 1567-1586 3′-UTR 16706TCA-CCT-GCA-ACA-ACC-CAG-GG 59 1604-1623 3′-UTR 16707GCG-GCT-AGT-CAC-CTG-CAA-CA 60 1612-1631 3′-UTR 16708CGC-TGG-GTT-TCG-CAG-GCA-GG 61 1631-1650 3′-UTR 16709ATC-ATC-TCC-TGA-AGA-ACG-CT 62 1647-1666 3′-UTR ¹Emboldened residues are2′-methoxyethoxy-modified.

TABLE 15 Rat and Mouse Gene Target Locations of JNK3 OligonucleotidesRat Mouse SEQ NUCLEOTIDE GENE NUCLEOTIDE GENE ISIS ID CO- TARGET CO-TARGET NO. NO: ORDINATES¹ REGION ORDINATES² REGION 16692 46 0213-02325′-UTR 0301-0320 tIR 16693 47 0222-0241 5′-UTR 0310-0329 tIR 16695 49 —— 0367-0386 ORF 16703 56 1506-1525 ORF 1571-1590 tTR 16704 57 1563-1582ORF 1628-1647 3′-UTR 16705 58 1588-1607 ORF 1653-1672 3′-UTR 16707 601633-1652 tTR 1698-1717 3′-UTR 16708 61 1652-1671 3′-UTR 1717-17363′-UTR ¹Co-ordinates from GenBank Accession No. L27128 (SEQ ID NO: 99),locus name “RATSAPKC.” ²Co-ordinates from GenBank Accession No. L35236(SEQ ID NO: 100), locus name “MUSMAPK.”

Oligonucleotides specific for JNK3 isoforms: Two isoforms of JNK3 havebeen described. JNK3-a1 was initially cloned and named “p49^(3F12)kinase” by (Mohit et al. Neuron, 1995, 14, 67). Subsequently, two cDNAsencoding related isoforms of JNK3 were cloned and their nucleotidesequences determined (Gupta et al., EMBO Journal, 1996, 15, 2760). Theisoforms are named JNK3-a1 (GenBank accession No. U34820 (SEQ ID NO:101), locus name “HSU34820”) and JNK3-a2 (GenBank accession No. U34819(SEQ ID NO: 102), locus name “HSU34819”) herein. The two isoforms ofJNK3, which probably arise from alternative mRNA splicing, can eachinteract with different transcription factors or sets of transcriptionfactors (Gupta et al., EMBO Journal, 1996, 15, 2760). As detailed below,certain oligonucleotides of the invention are specific for each of theseisoforms of JNK3.

JNK3-a1 and JNK-a2 differ at their carboxyl terminal portions. Thesubstantial differences in the amino acid sequences of these isoforms (5amino acids in JNK3-a1 are replaced with 47 amino acids in JNK3-a2)result from a slight difference in nucleotide sequence that shifts thereading frame. Specifically, in the ORF of mRNAs encoding JNK3-a1,nucleotides (nt) 1325-1362 of JNK3-a1 (GenBank accession No. U34820 (SEQID NO: 101)) have the sequence shown below as SEQ ID NO: 83, whereas, inthe ORF of mRNAs encoding JNK3-a2, nt 1301-1333 of JNK3-a2 (GenBankaccession No. U34819 (SEQ ID NO: 102)) have the sequence shown below asSEQ ID NO: 84. For purposes of illustration, SEQ ID NOS: 83 and 202 areshown aligned with each other (vertical marks, “|,” indicate bases thatare identical in both sequences; dashes, “-,” indicate bases that areabsent in the indicated sequence; and emboldened bases indicate the stopcodon for the JNK3-a1 ORF):

Due to this divergence between the JNK3 isoforms, antisenseoligonucleotides derived from the reverse complement of SEQ ID NO: 83(i.e., SEQ ID NO: 85, see below) are specifically hybridizable to mRNAsencoding JNK3-a1, and can be selected and used to modulate theexpression of JNK3-a1 without significantly effecting the expression ofJNK3-a2. In like fashion, antisense oligonucleotides derived from thereverse complement of SEQ ID NO: 84 (i.e., SEQ ID NO: 86, see below) arespecifically hybridizable to mRNAs encoding JNK3-a2, and can be selectedand used to modulate the expression of JNK3-a2 without significantlyeffecting the expression of JNK3-a1:

In preferred embodiments, such isoform-specific oligonucleotides such asare described above are methoxyethoxy “gapmers” or “wingmers” in whichthe RNase H-sensitive “gap” or “wing” is positioned so as to overlap aregion of nonidentity in the above antisense sequences, i.e., SEQ IDNOS: 85 and 86.

Activities of JNK3 oligonucleotides: The JNK3-specific phosphorothioate,2′-methoxyethoxy “gapmer” oligonucleotides (Table 14) were screened fortheir ability to affect JNK3 mRNA levels in SH-SYSY cells (Biedler etal., Cancer Res., 1973, 33, 2643). SH-SY5Y cells express a variety ofmitogen-activated protein kinases (MAPKs; see, e.g., Cheng et al., J.Biol. Chem., 1998, 273, 14560). Cells were grown in DMEM essentially aspreviously described (e.g., Singleton et al., J. Biol. Chem., 1996, 271,31791; Jalava et al., Cancer Res., 1990, 50, 3422) and treated witholigonucleotides at a concentration of 200 nM as described in Example 2.Control cultures were treated with an aliquot of LIPOFECTIN™ thatcontained no oligonucleotide.

The results are shown in Table 16. Oligonucleotides showing levels ofinhibition of JNK3 mRNA levels of at least 45% include ISIS Nos. 16692,16693, 16694, 16695, 16696, 16697, 16702, 16703, 16704, 16705 and 16706(SEQ ID NOS:46, 47, 48, 49, 50, 51, 55, 56, 57, 58 and 59,respectively). These oligonucleotides are preferred embodiments of theinvention for modulating JNK3 expression. Oligonucleotides inhibitingJNK3 mRNAs by at least 60% in this assay include ISIS Nos. 16693, 16702,16703 and 16704 (SEQ ID NOS: 47, 55, 56 and 57, respectively). Theseoligonucleotides are thus more preferred embodiments of the inventionfor modulating JNK3 expression.

TABLE 16 Activities of JNK3 Oligonucleotides SEQ ID GENE TARGET %ISISNo: NO: REGION EXPRESSION: % INHIBITION: control¹ — — 100% 0% 1669044 5′-UTR 60% 40% 16691 45 5′-UTR 66% 34% 16692 46 tIR 47% 53% 16693 47tIR 40% 60% 16694 48 ORF 42% 58% 16695 49 ORF 44% 56% 16696 50 ORF 55%45% 16697 51 ORF 54% 46% 16698 52 ORF 63% 37% 16699 53 ORF 61% 39% 1670054 ORF N.D.² N.D. 16702 55 ORF 39% 61% 16703 56 tTR 30% 70% 16704 573′-UTR 36% 64% 16705 58 3′-UTR 42% 58% 16706 59 3′-UTR 45% 55% 16707 603′-UTR 73% 27% 16708 61 3′-UTR 68% 32% 16709 62 3′-UTR 66% 34% ¹Cellstreated with LIPOFECTIN ™ only (no oligonucleotide). ²N.D., notdetermined.

Example 6 Oligonucleotides Targeted to Genes Encoding Rat JNK Proteins

In order to study the role of JNK proteins in animal models,oligonucleotides targeted to the genes encoding JNK1, JNK2 and JNK3 ofRattus norvegicus were prepared. These oligonucleotides are2′-methoxyethoxy, phosphodiester/2′-hydroxyl,phosphorothioate/2′-methoxyethoxy, phosphodiester “gapmers” in whichevery cytosine residue is 5-methylcytosine (m5c). These antisensecompounds were synthesized according to the methods of the disclosure.Certain of these oligonucleotides are additionally specificallyhybridizable to JNK genes from other species as indicated herein. Theoligonucleotides described in this Example were tested for their abilityto modulate rat JNK mRNA levels essentially according to the methodsdescribed in the preceding Examples, with the exceptions that the cellline used was rat A10 aortic smooth muscle cells (ATCC No. ATCCCRL-1476) and the probes used were specific for rat JNK1, JNK2 or JNK3(see infra). A10 cells were grown and treated with oligonucleotidesessentially as described by (Cioffi et al. Mol. Pharmacol., 1997, 51,383).

JNK1: Table 17 describes the sequences and structures of a set ofoligonucleotides, ISIS Nos. 21857 to 21870 (SEQ ID NOS:111 to 124,respectively) that were designed to be specifically hybridizable tonucleic acids from Rattus norvegicus that encode a stress-activatedprotein kinase named “p54?” or “SAPK?” that is homologous to the humanprotein JNK1 (Kyriakis et al., Nature, 1994, 369, 156; GenBank accessionNo. L27129 (SEQ ID NO: 88), locus name “RATSAPKD”). In Table 17,emboldened residues are 2′-methoxyethoxy-residues (others are2′-deoxy-); “C” residues are 2′-methoxyethoxy-5-methyl-cytosines and “C”residues are 5-methyl-cytosines; “o” indicates a phosphodiester linkage;and “s” indicates a phosphorothioate linkage. The target geneco-ordinates are from GenBank Accession No. L27129 (SEQ ID NO: 88),locus name “RATSAPKD.”

TABLE 17 Nucleotide Sequences of Rat JNK1 Oligonucleotides TARGET GENESEQ NUCLEOTIDE GENE ISIS ID CO- TARGET NO. NUCLEOTIDE SEQUENCE(5′ -> 3′)NO ORDINATE REGION 21857 CoAoAoCoGsTsCsCsCsGsCsGsCsTsCsGoGoCoCoG 1110002-0021 5′-UTR 21858 CoCoToGoCsTsCsGsCsGsGsCsTsCsCsGoCoGoToT 1120029-0048 5′-UTR 21859 CoToCoAoTsGsAsTsGsGsCsAsAsGsCsAoAoToToA 1130161-0180 tIR 21860 ToGoToToGsTsCsAsCsGsTsTsTsAsCsToToCoToG 1140181-0200 ORF 21861 CoGoGoToAsGsGsCsTsCsGsCsTsTsAsGoCoAoToG 1150371-0390 ORF 21862 CoToAoGoGsGsAsTsTsTsCsTsGsTsGsGoToGoToG 1160451-0470 ORF 21863 CoAoGoCoAsGsAsGsTsGsAsAsGsGsTsGoCoToToG 1170592-0611 ORF 21864 ToCoGoToTsCsCsTsGsCsAsGsTsCsCsToToGoCoC 1180691-0710 ORF 21865 CoCoAoToTsTsCsTsCsCsCsAsTsAsAsToGoCoAoC 1190811-0830 ORF 21866 ToGoAoAoTsTsCsAsGsGsAsCsAsAsGsGoToGoToT 1200901-0920 ORF 21867 AoGoCoToTsCsGsTsCsTsAsCsGsGsAsGoAoToCoC 1211101-1120 ORF 21868 CoAoCoToCsCsTsCsTsAsTsTsGsTsGsToGoCoToC 1221211-1230 ORF 21869 GoCoToGoCsAsCsCsTsAsAsAsGsGsAsGoAoCoGoG 1231301-1320 ORF 21870 CoCoAoGoAsGsTsCsGsGsAsTsCsTsGsToGoGoAoC 1241381-1400 ORFThese antisense compounds were tested for their ability to modulatelevels of (JNK1) and (JNK2) mRNA in A10 cells via Northern assays. Dueto the high degree of sequence identity between the human and rat genes,radiolabeled human JNK1 (Example 3) and JNK2 (Example 4) cDNAsfunctioned as specific probes for the rat homologs.

The results are shown in Table 18. ISIS Nos. 21857 to 21870 (SEQ IDNOS:111 to 124, respectively) showed 70% to 90% inhibition of rat JNK1mRNA levels. These oligonucleotides are preferred embodiments of theinvention for modulating rat JNK1 expression. Oligonucleotides showinglevels of inhibition of at least 90% in this assay include ISIS Nos.21858, 21859, 21860, 21861, 21862, 21864, 21865, 21866 and 21867 (SEQ IDNOS:112, 113, 114, 115, 116, 118, 119, 120 and 121, respectively). Theseoligonucleotides are thus more preferred embodiments of the inventionfor modulating rat JNK1 expression. ISIS 21859 (SEQ ID NO:113) waschosen for use in further studies (infra).

Two of the oligonucleotides, ISIS Nos. 21861 and 21867 (SEQ ID NOS:115and 121, respectively) demonstrated a capacity to modulate both JNK1 andJNK2. Such oligonucleotides are referred to herein as “Pan JNK”antisense compounds because the term “Pan” is used in immunologicalliterature to refer to an antibody that recognizes, e.g., all isoformsof a protein or subtypes of a cell type. The Pan JNK oligonucleotidesare discussed in more detail infra.

In addition to being specifically hybridizable to nucleic acids encodingrat JNK1, some of the oligonucleotides described in Table 17 are alsospecifically hybridizable with JNK1-encoding nucleic acids from otherspecies. ISIS 21859 (SEQ ID NO:113) is complementary to bases 4 to 23 ofcDNAs encoding human JNK1a1 and JNK1β1 (i.e., GenBank accession Nos.L26318 (SEQ ID NO: 87) and U35004 (SEQ ID NO: 90), respectively). ISIS21862 (SEQ ID NO:116) is complementary to bases 294 to 313 of the humanJNK1a1 and JNK1β1 cDNAs (GenBank accession Nos. L26318 (SEQ ID NO: 87)and U35004 (SEQ ID NO: 90), respectively), bases 289 to 308 of the humanJNK1132 cDNA (GenBank accession No. U35005 (SEQ ID NO: 91)), and bases288 to 307 of the human JNK1a2 cDNA (GenBank accession No. U34822 (SEQID NO: 89)). Finally, ISIS 21865 is complementary to bases 654 to 673 ofthe human JNK1a1 cDNA (GenBank accession No. L26318 (SEQ ID NO: 87)) andto bases 648 to 667 of the human JNK1a2 cDNA (GenBank accession No.U34822 (SEQ ID NO: 89)). These oligonucleotides are tested for theirability to modulate mRNA levels of human JNK1 genes according to themethods described in Example 3.

TABLE 18 Activities of Oligonucleotides Targeted to Rat JNK1 SEQ ID GENETARGET % EXPRESSION % EXPRESSION ISISNo: NO: REGION JNK1 JNK2 control¹ —— 100% 100% 21857 111 5′-UTR 24% 91% 21858 112 5′-UTR 8% 89% 21859 113tIR 5% 106% 21860 114 ORF 8% 98% 21861 115 ORF 6% 13% 21862 116 ORF 6%133% 21863 117 ORF 24% 107% 21864 118 ORF 8% 106% 21865 119 ORF 5% 50%21866 120 ORF 8% 98% 21867 121 ORF 5% 21% 21868 122 ORF 15% 112% 21869123 ORF 30% 93% 21870 124 ORF 11% 87% ¹Cells treated with LIPOFECTIN ™only (no oligonucleotide).

JNK2: Table 19 describes the sequences and structures of a set ofoligonucleotides, ISIS Nos. 18254 to 18267 (SEQ ID NOS:125 to 138,respectively) that were designed to be specifically hybridizable tonucleic acids that encode a stress-activated protein kinase from Rattusnorvegicus that encode a stress-activated protein kinase named “p54a” or“SAPKa” (Kyriakis et al., Nature, 1994, 369, 156). The structures ofthree control oligonucleotides, ISIS Nos. 21914 to 21916 (SEQ ID NOS:139to 141, respectively) are also shown in the table. Two isoforms of p54ahave been described: “p54a1” (GenBank accession No. L27112 (SEQ ID NO:93), locus name “RATSAPKA”) and “p54a2” (GenBank accession No. L27111(SEQ ID NO: 104), locus name “RATSAPKB”). With the exception of ISIS18257 (SEQ ID NO:128), the oligonucleotides described in Table 19 arespecifically hybridizable to nucleic acids encoding either p54a1 orp54a2. ISIS 18257 is specifically hybridizable to nucleic acids encodingp54a2 (i.e., GenBank accession No. L27112 (SEQ ID NO: 93), locus name“RATSAPKB”). In Table 19, emboldened residues are2′-methoxyethoxy-residues (others are 2′-deoxy-); “C” residues are2′-methoxyethoxy-5-methyl-cytosines and “C” residues are5-methyl-cytosines; “o” indicates a phosphodiester linkage; and “s”indicates a phosphorothioate linkage. The target gene co-ordinates arefrom GenBank Accession No. L27112 (SEQ ID NO: 93), locus name“RATSAPKB.”

TABLE 19 Nucleotide Sequences of Rat JNK2 Oligonucleotides SEQTARGET GENE GENE ISIS ID NUCLEOTIDE TARGET NO. NUCLEOTIDE SEQUENCE (5′-> 3′) NO: CO-ORDINATES REGION 18254 ToCoAoToGsAsTsGsTsAsGsTsGsTsCs 1250001-0020 tIR AoToAoCoA 18255 ToGoToGoGsTsGsTsGsAsAsCsAsCsAs 1260281-0300 ORF ToToToAoA 18256 CoCoAoToAsTsGsAsAsTsAsAsCsCsTs 1270361-0380 ORF GoAoCoAoT 18257 GoAoToAoTsCsAsAsCsAsTsTsCsTsCs 1280621-0640 ORF CoToToGoT 18258 GoCoToToCsGsTsCsCsAsCsAsGsAsGs 1290941-0960 ORF AoToCoCoG 18259 GoCoToCoAsGsTsGsGsAsCsAsTsGsGs 1301201-1220 ORF AoToGoAoG 18260 AoToCoToGsCsGsAsGsGsTsTsTsCsAs 1311281-1300 tTR ToCoGoGoC 18261 CoCoAoCoCsAsGsCsTsCsCsCsAsTsGs 1321341-1360 3′-UTR ToGoCoToC 18262 CoAoGoToTsAsCsAsCsAsTsGsAsTsCs 1331571-1590 3′-UTR ToGoToCoA 18263 AoAoGoAoGsGsAsTsTsAsAsGsAsGsA 1341701-1720 3′-UTR sToToAoToT 18264 AoGoCoAoGsAsGsTsGsAsAsAsTsAsC 1352001-2020 3′-UTR sAoAoCoToT 18265 ToGoToCoAsGsCsTsCsTsAsCsAsTsTs 1362171-2190 3′-UTR AoGoGoCoA 18266 AoGoToAoAsGsCsCsCsGsGsTsCsTsCs 1372371-2390 3′-UTR CoToAoAoG 18267 AoAoAoToGsGsAsAsAsAsGsGsAsCsA 1382405-2424 3′-UTR sGoCoAoGoC 21914 GoCoToCoAsGsTsGsGsAsTsAsTsGsGs 13918259 control — AoToGoAoG 21915 GoCoToAoAsGsCsGsGsTsCsAsAsGsG 14018259 control — sToToGoAoG 21916 GoCoToCoGsGsTsGsGsAsAsAsTsGsG 14118259 control — sAoToCoAoG

TABLE 20 Activities of Oligonucleotides Targeted to Rat JNK2 SEQ ID GENETARGET % ISISNo: NO: REGION EXPRESSION % INHIBITION control¹ — — 100% 0%18254 125 tIR 20% 80% 18255 126 ORF 21% 79% 18256 127 ORF 80% 20% 18257128 ORF 32% 68% 18258 129 ORF 19% 81% 18259 130 ORF 15% 85% 18260 131ORF 41% 59% 18261 132 3′-UTR 47% 53% 18262 133 3′-UTR 50% 50% 18263 1343′-UTR 63% 37% 18264 135 3′-UTR 48% 52% 18265 136 3′-UTR 38% 62% 18266137 3′-UTR 66% 34% 18267 138 3′-UTR 84% 16% ¹Cells treated withLIPOFECTIN ™ only (no oligonucleotide).

These antisense compounds were tested for their ability to modulatelevels of p54a (JNK2) mRNA in A10 cells using the radiolabeled humanJNK2 cDNA as a probe as described supra. The results are shown in Table20. Oligonucleotides showing levels of inhibition from ≧about 60% toabout 100% of rat JNK2 mRNA levels include ISIS Nos. 18254, 18255,18257, 18258, 18259, 18260 and 18265 (SEQ ID NOS:125, 126, 128, 129,130, 131 and 136, respectively). These oligonucleosides are preferredembodiments of the invention for modulating rat JNK2 expression.Oligonucleotides showing levels of inhibition of rat JNK1 mRNAs by atleast 80% in this assay include ISIS Nos. 18254, 18255, 18258 and 18259(SEQ ID NOS:125, 126, 129 and 130, respectively). These oligonucleotidesare thus more preferred embodiments of the invention for modulating ratJNK2 expression. ISIS 18259 (SEQ ID NO:130) was chosen for use infurther studies (infra).

Dose Response: A dose response study was conducted usingoligonucleotides targeted to rat JNK1 (ISIS 21859; SEQ ID NO:113) andJNK2 (ISIS 18259; SEQ ID NO:130) and Northern assays. The results (Table21) demonstrate an increasing effect as the oligonucleotideconcentration is raised and confirm that ISIS Nos. 21859 and 18259 (SEQID NOS:113 and 130, respectively) specifically modulates levels of mRNAencoding JNK1 and JNK2, respectively.

TABLE 21 Dose-Dependent Response to Rat JNK Antisense Oligonucleotides(ASOs) SEQ % EXPRES- % EXPRES- ID SION SION ISIS # NO: ASODescriptionDose JNK1 JNK2 21859 113 rat JNK1  0 nM 100 100 active ASO  10 nM 74 101 50 nM 25 98 100 nM 11 99 200 nM 8 101 18259 130 rat JNK2  0 nM 100 100active ASO  10 nM 95 81  50 nM 101 35 100 nM 94 15 200 nM 89 5

JNK3: Table 22 describes the sequences and structures of a set ofoligonucleotides, ISIS Nos. 21899 to 21912 (SEQ ID NOS:142 to 155,respectively) that were designed to be specifically hybridizable tonucleic acids from Rattus norvegicus that encode a stress-activatedprotein kinase named “p54B” that is homologous to the human protein JNK3(Kyriakis et al., Nature, 1994, 369, 156; GenBank accession No. L27128(SEQ ID NO: 99), locus name “RATSAPKC”). In Table 22, emboldenedresidues are 2′-methoxyethoxy-residues (others are 2′-deoxy-); “C”residues are 2′-methoxyethoxy-5-methyl-cytosines and “C” residues are5-methyl-cytosines; “o” indicates a phosphodiester linkage; and “s”indicates a phosphorothioate linkage. The target gene co-ordinates arefrom GenBank Accession No. L27128 (SEQ ID NO: 99), locus name“RATSAPKC.” The oligonucleotides are tested for their ability tomodulate rat JNK3 mRNA levels essentially according to the methodsdescribed in the preceding Examples.

In addition to being specifically hybridizable to nucleic acids encodingrat JNK3, some of the oligonucleotides described in Table 22 are alsospecifically hybridizable with JNK3-encoding nucleic acids from humansand Mus musculus (mouse). Table 23 sets out these relationships. Theseoligonucleotides are tested for their ability to modulate mRNA levels ofthe human JNK genes according to the methods described in Example 5.

TABLE 22 Nucleotide Sequences of Rat JNK3 Oligonucleotides TARGET GENESEQ NUCLEOTIDE GENE ISIS NUCLEOTIDE SEQUENCE ID CO- TARGET NO. (5′ ->3′) NO: ORDINATES REGION 21899 GoGoGoCoTsTsTsCsAsTsTsAs 142 0021-00405′-UTR GsCsCsAoCoAoToT 21900 GoGoToToGsGsTsTsCsAsCsTs 143 0241-02605′-UTR GsCsAsGoToAoGoT 21901 ToGoCoToCsAsTsGsTsTsGsTs 144 0351-0370 tIRAsAsTsGoToToToG 21902 GoToCoGoAsGsGsAsCsAsGsCs 145 0491-0510 ORFGsTsCsAoToAoCoG 21903 CoGoAoCoAsTsCsCsGsCsTsCs 146 0731-0750 ORFGsTsGsGoToCoCoA 21904 AoCoAoToAsCsGsGsAsGsTsCs 147 0901-0920 ORFAsTsCsAoToGoAoA 21905 GoCoAoAoTsTsTsCsTsTsCsAsT 148 1101-1120 ORFsGsAsAoToToCoT 21906 ToCoGoToAsCsCsAsAsAsCsGs 149 1321-1340 ORFTsTsGsAoToGoToA 21907 CoGoCoCoGsAsGsGsCsTsTsCs 150 1601-1620 ORFCsAsGsGoCoToGoC 21908 GoGoCoToAsGsTsCsAsCsCsTs 151 1631-1650 tTRGsCsAsAoCoAoAoC 21909 GoCoGoToGsCsGsTsGsCsGsTs 152 1771-1790 3′-UTRGsCsTsToGoCoGoT 21910 GoCoToCoAsGsCsTsGsCsGsAs 153 1891-1910 3′-UTRTsAsCsAoGoAoAoC 21911 AoGoCoGoCsGsAsCsTsAsGsAs 154 1921-1940 3′-UTRAsGsTsToAoAoGoT 21912 AoGoGoGoAsGsAsCsCsAsAsAs 155 1941-1960 3′-UTRGsTsCsGoAoGoCoG

TABLE 23 Cross-Hybridizations of Rat JNK3 Oligonucleotides ISIS SEQ IDHybridizes to: NO. NO: Human JNK3a1¹ Human JNK3a2² MouseJNK3³ 21900 143— — bp 329-348 21901 144 bp 193-212 bp 169-188 bp 411-430 21904 147 — —bp 961-980 21905 148 bp 943-962 bp 919-938 — 21906 149 — — bp 1381-140021908 151 bp 1478-1497 bp 1449-1468 bp 1696-1715 ¹GenBank accession No.U34820 (SEQ ID NO: 101), locus name “HSU34820” (see also Mohit et al.,Neuron, 1995, 14, 67 and Gupta et al., EMBO Journal, 1996, 15, 2760).²GenBank accession No. U34819 (SEQ ID NO: 102), locus name “HSU34819”(see also Gupta et al., EMBO Journal, 1996, 15, 2760). ³Also known asp459^(3F12) MAPK; GenBank accession No. L35236 (SEQ ID NO: 100), locusname “MUSMAPK” (see also Martin et al., Brain Res. Mol. Brain Res.,1996, 35, 47).

Pan JNK Oligonucleotides: Certain of the oligonucleotides of theinvention are capable of modulating two or more JNK proteins and arereferred to herein as “Pan JNK” oligonucleotides. For example, ISIS Nos.21861 and 21867 (SEQ ID NOS:115 and 121, respectively) demonstrated acapacity to modulate both JNK1 and JNK2 (Table 18). Sucholigonucleotides are useful when the concomitant modulation of severalJNK proteins is desired.

Human Pan JNK oligonucleotides are described in Table 24. Theseoligonucleotides are designed to be complementary to sequences that areidentically conserved in (i.e., SEQ ID NOS:156, 158, 159, 160 and 161),or which occur with no more than a one-base mismatch (SEQ ID NO:157), innucleic acids encoding human JNK1a1, JNK1a2, JNK2a1 and JNK2a2. Theoligonucleotides described in Table 24 are evaluated for their

ability to modulate JNK1 and JNK2 mRNA levels in A549 cells using themethods and assays described in Examples 3 and 4.

In instances where such common sequences encompass one or more basedifferences between the JNK genes that it is desired to modulate,hypoxanthine (inosine) can be incorporated at the positions of theoligonucleotide corresponding to such base differences. (“Hypoxanthine”is the art-accepted term for the base that corresponds to the nucleosideinosine; however, the term “inosine” is used herein in accordance withU.S. and PCT rules regarding nucleotide sequences.) As is known in theart, inosine (I) is capable of hydrogen bonding with a variety ofnucleobases and thus serves as a “universal” base for hybridizationpurposes. For example, an oligonucleotide having a sequence that is aderivative of SEQ ID NO:157 having one inosine substitution(TAGGAIATTCTTTCATGATC, SEQ ID NO:162) is predicted to bind to nucleicacids encoding human JNK1a1, JNK1a2, JNK2a1 and JNK2a2 with nomismatched bases. As another example, an oligonucleotide having asequence that is a derivative of SEQ ID NO:161 having one inosinesubstitution (GGTTGCAITTTCTTCATGAA, SEQ ID NO:163) is predicted to bindwith no mismatched bases to nucleic acids encoding human JNK3a1 andJNK3a2 in addition to JNK1a1, JNK1a2, JNK2a1 and JNK2a2. Sucholigonucleotides are evaluated for their ability to modulate JNK1 andJNK2 mRNA levels in A549 cells, and JNK3 mRNA levels in SH-SY5Y cells,using the methods and assays described in Examples 3, 4 and 5.

TABLE 24 Human Pan INK Oligonucleotides NUCLEOTIDE SEQUENCE (5′ -> 3′)SEQ AND CHEMICAL MODIFICATIONS* ID NO: A ^(S) C ^(S) A ^(S) T ^(S) C^(S) T ^(S)T^(O)G^(O)A^(O)A^(O)A^(O)T^(O)T^(O)C^(S) T ^(S) T ^(S) C ^(S)T ^(S) A ^(S) G 156 T ^(S) A ^(S) G ^(S) G ^(S) A ^(S) T^(S)A^(O)T^(O)T^(O)C^(O)T^(O)T^(O)T^(O)C^(S) A ^(S) T ^(S) G ^(S) A ^(S)T ^(S) C 157 A ^(S) G ^(S) A ^(S) A ^(S) G ^(S) G^(S)T^(O)A^(O)G^(O)G^(O)A^(O)C^(O)A^(O)T^(S) T ^(S) C ^(S) T ^(S) T ^(S)T ^(S) C 158 T ^(S) T ^(S) T ^(S) A ^(S) T ^(S) T^(S)C^(O)C^(O)A^(O)C^(O)T^(O)G^(O)A^(O)T^(S) C ^(S) A ^(S) A ^(S) T ^(S)A ^(S) T 159 T ^(S) C ^(S) A ^(S) A ^(S) T ^(S) A^(S)A^(O)C^(O)T^(O)T^(O)T^(O)A^(O)T^(O)T^(S) C ^(S) C ^(S) A ^(S) C ^(S)T ^(S) G 160 G ^(S) G ^(S) T ^(S) T ^(S) G ^(S) C^(S)A^(O)G^(O)T^(O)T^(O)T^(O)C^(O)T^(O)T^(S) C ^(S) A ^(S) T ^(S) G ^(S)A ^(S) A 161 *Emboldened residues, 2′-methoxyethoxy-residues (others are2′-deoxy-); all “C” residues are 5-methyl-cytosines; “^(O)”,phosphodiester linkage; “^(S)”, phosphorothioate linkage.

Example 7 Effect of Oligonucleotides Targeted to Human JNK1 and JNK2 onTNFa-induced JNK Activity

Human umbilical vein endothelial cells (HUVEC, Clonetics, San DiegoCalif.) were incubated with oligonucleotide with LipofectinJ inOpti-MEMJ for 4 hours at 37° C./5% CO₂. The medium was then replacedwith 1% FBS/EGM (Clonetics, Walkersville Md.) and incubated for 24 hoursat 37° C./5% CO₂. Cells were treated with 5 ng/ml TNFa for 15 minutesbefore lysis. JNK activity was determined by incubating lysates(normalized for protein) with immobilized GST-c-Jun fusion protein(e.g., New England Biolabs, Beverly, Mass.)+³²P-ATP. GST-c-Jun beadswere washed and SDS-PAGE sample buffer was added. Samples were resolvedby SDS-PAGE and phosphorylated c-Jun was visualized using a MolecularDynamics PhosphorImager.

Compared to a control oligonucleotide, the JNK1 oligonucleotide ISIS15346 (SEQ ID NO: 16; 100 nM concentration) inhibited TNFa-induced JNKactivity by approximately 70%. The JNK2 oligonucleotide ISIS 15353 (SEQID NO: 31; 100 nM) inhibited TNFa-induced JNK activity by approximately55%. A combination of 50 nM each oligonucleotide inhibited TNFa-inducedJNK activity by approximately 68% and a combination of 100 nM eacholigonucleotide inhibited TNFa-induced JNK activity by approximately83%.

Example 8 Inhibition of Inflammatory Responses by AntisenseOligonucleotides Targeting JNK Family Members

JNKs have been implicated as key mediators of a variety of cellularresponses and pathologies. JNKs can be activated by environmentalstress, such as radiation, heat shock, osmotic shock, or growth factorwithdrawal as well as by pro-inflammatory cytokines.

Antisense oligonucleotides targeting any of the JNK family membersdescribed in Examples 3-5 are synthesized and purified as in Example 1and evaluated for their activity in inhibiting inflammatory responses.Such inhibition is evident in the reduction of production ofpro-inflammatory molecules by inflammatory cells or upon the attenuationof proliferation of infiltrating or inflammatory cells, the mostprominent of which are lymphocytes, neutrophils, macrophages andmonocytes. Following synthesis, oligonucleotides are tested in anappropriate model system using optimal tissue or cell cultureconditions. Inflammatory cells including lymphocytes, neutrophils,monocytes and macrophages are treated with the antisenseoligonucleotides by the method of electroporation. Briefly, cells (5×10⁶cells in PBS) are transfected with oligonucleotides by electroporationat 200V, 1000 uF using a BTX Electro Cell Manipulator 600 (Genetronics,San Diego, Calif.). For an initial screen, cells are electroporated with10 uM oligonucleotide and RNA is collected 24 hours later. Controlswithout oligonucleotide are subjected to the same electroporationconditions.

Total cellular RNA is then isolated using the RNEASY7 kit (Qiagen, SantaClarita, Calif.). RNAse protection experiments are conducted usingRIBOQUANT™ kits and template sets according to the manufacturer'sinstructions (Phaimingen, San Diego, Calif.).

Adherent cells such as endothelial and A549 cells are transfected usingthe LIPOFECTIN™ protocol described in Example 2. Reduced JNK mRNAexpression is measured by Northern analysis while protein expression ismeasured by Western blot analysis, both described in Example 1. Negativecontrol oligonucleotides with mismatch sequences are used to establishbaselines and non-specific effects.

The degree of inflammatory response is measured by determining thelevels of inflammatory cytokine expression by Northern or Westernanalysis, or cytokine secretion by enzyme-linked immunosorbent assay(ELISA) techniques. Enzyme-linked immunosorbent assays (ELISA) arestandard in the art and can be found at, for example, Ausubel, F. M. etal., Current Protocols in Molecular Biology, Volume 2, pp.11.2.1-11.2.22, John Wiley & Sons, Inc., 1991.

The degree of inflammatory response is also determined by measuring theexpression of known immediate-early genes by the method of Northern orWestern blot analysis. Further into the inflammatory response, levels ofapoptosis are measured by flow cytometry.

Example 9 Inhibition of Fibrosis by Antisense Oligonucleotides TargetingJNK Family Members

Pulmonary fibrosis is characterized by inflammatory andfibroproliferative changes in the lung and an excess accumulation ofcollagen in the interstitium. There is also an increased recruitment ofimmune and inflammatory cells to the lung which act not only in theinitial damage to the lung but in the progression of the fibroticprocess.

In the rodent bleomycin (BL)-induced pulmonary fibrosis model,inhibition of fibrosis in the lung is determined by measuring any ofseveral markers for the condition. The BL-induced model is widelyaccepted in the art and can be found at, for example, Thrall, R. S. etal., Bleomycin In: Pulmonary Fibrosis, pp. 777-836, Eds. Phan, S. H. andThrall, R. S., Marcel Dekker, New York, 1995 and Giri, S, N. et al.,Miscellaneous mediator systems in pulmonary fibrosis In: PulmonaryFibrosis, pp. 231-292, Eds. Phan, S. H. and Thrall, R. S., MarcelDekker, New York, 1995.

Antisense oligonucleotides targeting any of the JNK family membersdescribed in Examples 3-5 are synthesized and purified as in Example 1and evaluated for their ability to prevent or inhibit pulmonaryfibrosis. These fibrotic markers include release of variouspro-inflammatory mediators including cytokines and chemokines such asTNFa, interleukin-8 and interleukin-6, increased numbers of proteasesand metalloproteinases, generation of reactive oxygen species (ROS),edema, hemorrhage and cellular infiltration predominated by neutrophilsand macrophages.

Following synthesis, oligonucleotides are tested in the rodentBL-induced pulmonary fibrosis model using optimal conditions. Micereceive an intratracheal dose of bleomycin (0.125 U/mouse) or saline,followed by treatment with antisense oligonucleotide (i.p.) over 2weeks. After 2 weeks mice are sacrificed and biochemical,histopathological and immunohistochemical analyses are performed.

Biochemical and immunohistochemical analysis involves the measurement ofthe levels of pro-inflammatory cytokine expression by Northern orWestern analysis, or cytokine secretion by enzyme-linked immunosorbentassay (ELISA) techniques as described in Example 8. Histopathologicalanalyses are performed for the presence of fibrotic lesions in theBL-treated lungs and for the presence of and number of cells with thefibrotic phenotype by methods which are standard in the art.

Example 10 Sensitization to Chemotherapeutic Agents by AntisenseOligonucleotides Targeting JNK Family Members

Manipulation of cancer chemotherapeutic drug resistance can also beaccomplished using antisense oligonucleotides targeting JNK familymembers. Antisense oligonucleotides targeting any of the JNK familymembers described in Examples 3-5 are synthesized and purified as inExample 1 and evaluated for their ability to sensitize cells to theeffects of chemotherapeutic agents. Sensitization is evident in theincreased number of target cells undergoing apoptosis subsequent totreatment. Following synthesis, oligonucleotides are tested in anappropriate model system using optimal tissue or cell cultureconditions. Cells are treated with the compounds of the invention inconjunction with one or more chemotherapeutic agents in a treatmentregimen wherein the chemotherapeutic agents can be used individually(e.g., 5-FU and oligonucleotide), sequentially (e.g., 5-FU andoligonucleotide for a period of time followed by MTX andoligonucleotide), or in combination with one or more other suchchemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or 5-FU,radiotherapy and oligonucleotide).

For nonadherent cells, treatment is by the method of electroporation.Briefly, cells (5×10⁶ cells in PBS) are transfected witholigonucleotides by electroporation either before, during or aftertreament with the chemotherapeutic agent, at 200V, 1000 uF using a BTXElectro Cell Manipulator 600 (Genetronics, San Diego, Calif.). For aninitial screen, cells are electroporated with 10 uM oligonucleotide andRNA is collected 24 hours later. Controls without oligonucleotide orchemotherapeutic agent are subjected to the same electroporationconditions.

Total cellular RNA is then isolated using the RNEASY7 kit (Qiagen, SantaClarita, Calif.). RNAse protection experiments are conducted usingRIBOQUANT™ kits and template sets according to the manufacturer'sinstructions (Pharmingen, San Diego, Calif.).

Adherent cells such as endothelial and A549 cells are transfected usingthe LIPOFECTIN™ protocol described in Example 2. Reduced JNK mRNAexpression is measured by Northern analysis while protein expression ismeasured by Western blot analysis, both described in Example 1. Negativecontrol oligonucleotides with mismatch sequences can be used toestablish baselines and non-specific effects. The degree of apoptosis,and consequently sensitization is measured by flow cytometry.

Example 11 Oligonucleotide-Mediated Inhibition of Human JNK2 ExpressionUsing a Cross-Species Oligonucleotide, ISIS 101759

In a further embodiment, chemical modifications to ISIS 18259 (SEQ IDNO: 130), designed to the rat JNK2 target were made and theoligonucleotide was investigated for activity in human cell lines.

The modified oligonucleotide, ISIS 101759, has identical base and sugarcompositions as ISIS 18259 and differs only in the linker composition.ISIS 101759 contains phosphorothioate linkages throughout. A comparisonof the two oligonucleotides is shown below.

“GoCoToCoAsGsTsGsGsAsCsAsTsGsGsAoToGoAoG” ISIS 18259“GsCsTsCsAsGsTsGsGsAsCsAsTsGsGsAsTsGsAsG” ISIS 101759

Both oligonucleotides have the following base sequence5′-GCTCAGTGGACATGGATGAG-3′ and emboldened residues are2′-methoxyethoxy-residues (others are 2′-deoxy-); “C” residues are2′-methoxyethoxy-5-methyl-cytosines and “C” residues are5-methyl-cytosines; “o” indicates a phosphodiester linkage; and “s”indicates a phosphorothioate linkage.

While ISIS 18259 was designed to target gene co-ordinates 1201-1220 fromGenBank Accession No. L27112 (SEQ ID NO: 93) (herein incorporated as SEQID NO: 168), locus name “RATSAPKB as dileneated in Table 19, this samesequence is also complementary over 18 of its 20 nucleobases tocoordinates 1248-1265 of human JNK2 from GenBank accession No. L31951(SEQ ID NO: 92) (herein incorporated as SEQ ID NO: 167), locus name“HUMJNK2”. The region of complementarity between ISIS 18259 (andconsequently 101759 since it has the same base sequence as ISIS 18259)and the human gene is shown here in bold, 5′-GCTCAGTGGACATGGATGAG-3′. Infact it is only the two nucleobases at the 3′ end of the oligonucleotidethat are not complementary to the human JNK2 gene.

Using three human cell lines, ISIS 101759 (SEQ ID NO: 130) was testedfor its ability to reduce human JNK2 RNA levels. The controloligonucleotide for the three studies was ISIS 101760 (SEQ ID NO: 166; a7-base mismatch). The control oligonucleotide has the same sugar andlinker sequence as ISIS 101759 and the nucleobase sequence,5′GsCsAsCsAsTsTsGsCsAsCsGsTsGsAsAsTsTsAsC-3′, where emboldened residuesare T-methoxyethoxy-residues (others are 2′-deoxy-); “C” residues are2′-methoxyethoxy-5-methyl-cytosines and “C” residues are5-methyl-cytosines; and “s” indicates a phosphorothioate linkage.

Inhibition of Human JNK2 in HuVEC Cells HuVEC Cells:

The human umbilical vein endothilial cell line HuVEC was obtained fromClonetics (Clonetics Corporation Walkersville, Md.). HuVEC cells wereroutinely cultured in EBM (Clonetics Corporation Walkersville, Md.)supplemented with SingleQuots supplements (Clonetics Corporation,Walkersville, Md.). Cells were routinely passaged by trypsinization anddilution when they reached 90% confluence were maintained for up to 15passages. Cells were seeded into 100 mm dishes and incubated overnightat 37° C./5% CO₂. (Falcon-Primaria #3872).

For Northern blotting or other analyses, cells can be seeded onto 100 mmor other standard tissue culture plates and treated similarly, usingappropriate volumes of medium and oligonucleotide. Treatment of HuVECcells with antisense compounds:

When cells reached 70% confluency, they were treated witholigonucleotide. For cells grown in 10 cm dishes, cells were washed oncewith 5 ml PBS and then treated with 5 ml of OPTI-MEM-1 containing 3 ulLIPOFECTIN (Invitrogen Corporation, Carlsbad, Calif.)/100 nMoligonucleotide/ml OPTI-MEM-1. For other oligonucleotide concentrationsthe oligonucleotide/Lipofectin ration was held constant. After 4-7 hoursof treatment, the medium was replaced with fresh medium. Cells wereharvested 16-24 hours after oligonucleotide treatment.

In accordance with the present invention, HuVEC cells were treated with100 nM ISIS 101759 or the control oligonucleotide and mRNA levels ofhuman JNK2 were monitored over a time-course of 0-72 hours andquantitated by Northern analysis. The data is shown in Table 25.

TABLE 25 Time-course Response to Rat JNK2 Antisense Oligonucleotides(ASOs) in HuVEC cells Percent Inhibition of human JNK2 mRNA ExpressionISIS Number 0 hr 12 hr 24 hr 48 hr 72 hr Control 0 6 7 23 16 101759 0 9392 88 70

From the data, it is evident that the rat JNK2 oligonucleotide wascapable of reducing the expression of human JNK2 in human HuVEC cells,and that by 72 hours the expression began to recover.

Inhibition of Human JNK2 in HeLa Cells HeLa Cells:

The human cervix epithelial adenocarcinoma cell line HeLa was obtainedfrom the American Type Culture Collection (Manassas, Va.). HeLa cellswere routinely cultured in Minimum essential medium (Eagle) with 2 mML-glutamine and Earle's BSS adjusted to contain 1.5 g/L sodiumbicarbonate, 0.1 mM non-essential amino acids, and 1.0 mM sodiumpyruvate, 90%; fetal bovine serum, 10% at a temperature of 37° C. Cellswere seeded into 100 mm dishes and incubated overnight at 37° C./5% CO₂

For Northern blotting or other analyses, cells can be seeded onto 100 mmor other standard tissue culture plates and treated similarly, usingappropriate volumes of medium and oligonucleotide.

Treatment of HeLa Cells with Antisense Compounds:

When cells reached 70% confluency, they were treated witholigonucleotide. For cells grown in 10 cm dishes, cells were washed oncewith 5 ml PBS and then treated with 5 ml of OPTI-MEM-1 containing 3 ulLIPOFECTIN (Invitrogen Corporation, Carlsbad, Calif.)/100 nMoligonucleotide/ml OPTI-MEM-1. For other oligonucleotide concentrationsthe oligonucleotide/Lipofectin ration was held constant. After 4-7 hoursof treatment, the medium was replaced with fresh medium. Cells wereharvested 16-24 hours after oligonucleotide treatment.

In accordance with the present invention, HeLa cells were treated with10, 50 or 200 nM ISIS 101759 or the control oligonucleotide and mRNAlevels of human JNK2 were quantitated by Northern analysis. The data isshown in Table 26.

TABLE 26 Dose Response to Rat JNK2 Antisense Oligonucleotides (ASOs) inHeLa cells Percent Inhibition of human JNK2 mRNA ISIS No: 10 nM 50 nM200 nM Control 0 0 1 101759 0 90 99

From the data, it is evident that the rat JNK2 oligonucleotide wascapable of reducing the expression of human JNK2 in human HeLa cells ina dose-dependent manner. HeLa cells were also treated with thetransfection reagent, lipofectamine, alone at 50 and 200 nM with noreduction in expression being observed.

Inhibition of Human JNK2 in Jurkat Cells Jurkat Cells:

The human Jurkat cell line was obtained from the American Type CultureCollection (ATCC) (Manassas, Va.). Jurkat cells were routinely culturedin RPMI Medium 1640 (Gibco/Life Technologies, Gaithersburg, Md.)supplemented with 20% fetal calf serum (Gibco/Life Technologies,Gaithersburg, Md.). Cells were routinely passaged by aspirating mediathat contained excess cells and replenishing with new media.

For electroporation, cells were diluted to 28×10⁶ cells/mL and placedinto 1 mm electroporation cuvettes. Electroporation is performed bytreating with 1-20 μM oligonucleotide, at 160 Volts for 6 msec. Theentire electroporated samples are then placed into 5 mL of 10% FBS/RPMIMedium 1640 in 100 mm plates. Plates are then left overnight at 37°C./5% CO₂.

Each sample is then transferred to 15 mL conical tubes and spun down at1200 rpm for 5 minutes followed by aspiration of the supernatant. Cellsare then suspended in 5 mL PBS followed by a second centrifugation at1200 rpm for 5 minutes followed by aspiration of the supernatant. Cellsare then washed and lysed. Following the lysis step, total cellular RNAis then isolated using the RNEASY kit (Qiagen, Santa Clarita, Calif.) asdescribed in other examples herein.

In accordance with the present invention, Jurkat cells were treated byelectroporation with 1, 5 or 20 uM ISIS 101759 or the controloligonucleotide and mRNA levels of human JNK2 were quantitated byNorthern analysis. The data is shown in Table 27.

TABLE 27 Dose Response to Rat JNK2 Antisense Oligonucleotides (ASOs) inJurkat cells Percent Inhibition of human JNK2 mRNA ISIS No: 1 uM 5 uM 20uM Control 12 18 19 101759 14 56 92

From the data, it is evident that the rat JNK2 oligonucleotide wascapable of reducing the expression of human JNK2 in human Jurkat cellsin a dose-dependent manner. Jurkat cells were also electroporated withreagents alone (no oligonucleotides) with no reduction in expressionbeing observed.

Targeting JNK1 for Metabolic Disorders Example 12 In Vivo Studies in anob/ob Model of Obesity

Leptin is a hormone produced by fat that regulates appetite.Deficiencies in this hormone in both humans and non-human animals leadsto obesity. ob/ob mice have a mutation in the leptin gene which resultsin obesity and hyperglycemia. As such, these mice are a useful model forthe investigation of obesity and metabolic syndrome and treatmentsdesigned to treat these conditions. ob/ob mice have higher circulatinglevels of insulin and are less hyperglycemic than db/db mice, whichharbor a mutation in the leptin receptor. In accordance with the presentinvention, the oligomeric compounds of the invention are tested in theob/ob model of obesity and diabetes.

Seven-week old male C57Bl/6J-Lep ob/ob mice (Jackson Laboratory, BarHarbor, Me.) were divided into a saline group (n=6), an oligonucleotidecontrol group (n=6) and a treatment group (n=6). All animals were fed adiet with a fat content of 10-15% for 6 weeks. The oligonucleotidecontrol group and the treatment group received a 25 mg/kg subcutaneousinjection of either the control oligonucleotide or the treatmentoligonucleotide twice a week for the 6 week perion. The saline groupreceived a saline injection on the same injection schedule. The controloligonucleotide in this study was a chimeric oligonucleotide that is 20nucleosides in length and is not targeted to a nucleic acid encodingJNK. (CCTTCCCTGAAGGTTCCTCC, SEQ ID NO: 107, (Isis No. 141923). Thetreatment oligonucleotide is also a chimeric oligonucleotide that is 20nucleotides in length, but it is targeted to a nucleic acid that encodesJNK1 polypeptide. The nucleic acid encoding JNK1 polypeptide has anucleoside sequence that is substantially similar to GenBank AccessionNo.: L27129 (SEQ ID NO: 88). 1; SEQ ID NO.: 88. The oligonucleotidecompound is also targeted to a nucleic acid that encodes mouse JNK1polypeptide with only a 1-nucleobase mismatch. The nucleic acidpreferably being substantially similar to GenBank Accession No.:NM_(—)016700.2; SEQ ID NO. 106. The treatment oligo contains thenucleobase sequence of TGTTGTCACGTTTACTTCTG, SEQ ID NO.: 114 (Isis No.104492). The oligonucleotide compound is also targeted to a nucleic acidthat encodes human JNK1 polypeptide with only a 2-nucleobase mismatch.The nucleic acid preferably being substantially similar to GenBankAccession NOs.: L26318 (SEQ ID NO: 87), U34822 (SEQ ID NO: 89), U35004(SEQ ID NO: 90) or U35005 (SEQ ID NO: 91); SEQ ID NOs.: 87, 89, 90, and91 respectively.

During the treatment period, weekly food intake was monitored, as werechanges in body weight. Body composition, blood biochemistry, metabolicrate, insulin tolerance, and oral glucose tolerance was also measured atcertain time points during the treatment period. After the treatmentperiod, mice were sacrificed and target mRNA levels were evaluated inliver, brown adipose tissue (BAT) and white adipose tissue (WAT), aswere histological, biochemical and molecular biology parameters. RNAisolation and target mRNA expression level quantitation are performed asdescribed by other examples herein unless otherwise stated.

mRNA Levels

Total RNA was isolated by homogenizing tissues in RLT buffer (Qiagen,Md.) followed by centrifugation with cesium chloride gradient. Real-timequantitative RT-PCR analysis was then performed to analyze the geneexpression.

JNK1, but not JNK 2 mRNA levels were reduced in the liver, white adiposetissue and brown adipiose tissue for the treatment group compared tosaline and oligonucleotide control groups(80% reduction in liver, 80%reduction in WAT and 78% reduction in BAT). There were no significantdifferences in JNK1 mRNA levels between the saline and oligonucleotidecontrol groups. Additionally, there were no significant differences inJNK2 mRNA levels between treatment, saline and oligonucleotide controlgroups. Thus, the treatment compound is specific for JNK1 over JNK2 inreducing mRNA expression.

JNK1 Activity Assay and Western Immunoblotting Analysis.

There was a corresponding reduction in JNK activity as determined byimmunoprecipitation using an antibody raised to JNK1 (Cell Signaling,Beverly, Mass.). JNK1 ASO resulted in a decrease in JNK1 activity bygreater than 95%, 80% and 65% in liver, WAT and BAT, respectively.

Feed Efficiency, Body Weight and Fat

As compared to control groups, the treatment group had an improved feedefficiency (change in body weight per volume of food intake). Thetreatment group feed efficiency was 0.052±0.0026 and the controloligonucleotide group was 0.060±0.002. The treatment group also showed areduction in body weight gain by about 20%, indicating an increasedmetabolic rate. Epididymal fat pad weight (3.8 g v. 4.5 g) and wholebody fat content (31.6% v. 35.5%) were also reduced. Indirectcalorimetry measurement confirmed that the treatment group had anincreased metabolic rate as reflected in a higher VO₂ as compared to theoligonucleotide control group (VO₂ increase over control group of >7%(greater than 7%) in the dark and 23% or greater in the light).

Glucose and Insulin Levels

Fed and fasting plasma glucose and plasma insulin levels were improvedfor the treatment group over the control group. (see Table 28). Glucoselevels were completely normalized and insulin levels were lowered bygreater than 50% after 6 weeks of treatment (Table 28) demonstratingincreased insulin sensitazation. Plasma glucose levels are measuredusing an Olympus Clinical Analysis (Olympus AU400, Olympus American Inc,Melville, N.Y.) and insulin levels are measure using an Alpcoinsulin-specific ELISA kit from (Windham, N.H.).

TABLE 28 Plasma Glucose and Insulin Levels in ob/ob Mice Saline ControlISIS 104492 Glucose Baseline 378.3 ± 33.0 374.2 ± 17.2 375.2 ± 33.7 (mg/dl) Fed 600.5 ± 48.7 445.5 ± 57.4  177.6 ± 12.3** Fasting 142.7 ±11.8 152.3 ± 23.5  90.6 ± 10.7* Insulin Baseline 30.2 ± 4.1 30.6 ± 3.729.8 ± 1.8  (ng/ml) Fed 24.4 ± 3.4 26.5 ± 5.8 11.0 ± 4.8* Fasting 17.6 ±1.8 16.9 ± 3.4  7.9 ± 0.4** Data are expressed as the mean ± SEM (n =5-6). *P < 0.05 and **P < 0.01 when compared to either control group.

To confirm this ASO-caused insulin-sensitizing effect, both Glucose(OGTT) and insulin tolerance tests (ITT) were administered in fed andfasted mice. Mice receive intraperitoneal injections of either glucoseor insulin, and the blood glucose and insulin levels are measured beforethe insulin or glucose challenge and at 15, 20 or 30 minute intervalsfor up to 2 hours. Blood glucose levels were measured using a Glucometer(Abbott Laboratories, Bedford, Mass.).

Insulin tolerance and oral glucose tolerance was improved for thetreatment group compared to the control groups. A glucose tolerance testin medical practice is the administration of glucose to determine howquickly it is cleared from the blood and is used to test for diabetes,insulin resistance, and sometimes reactive hypoglycemia. The results ofan oral glucose tolerance test of the mice of Example 12 are shown inTable 29.

TABLE 29 OGTT performed at 6 weeks (0.75 g/kg Glucose) Glucose mg/dL 0min 30 min 60 min 100 min Saline 150 425 320 375 Control 150 420 325 300104492 100 245 210 225

In response to glucose challenge, animals treated with JNK antisenseoligonucleotide show improved glucose tolerance. Peak plasma glucoselevel at the 30 minute time point was decreased by over 40% fromcontrols and the subsequent drop in glucose was lessened compared tocontrols. The AUC for glucose excursion was significantly lowered aftertreatment with JNK antisense oligonucleotide, indicating that inhibitionof JNK by antisense improves glucose tolerance. The results indicatethat glucose is cleared much more quickly from the blood of mice treatedwith JNK antisense oligonucleotide relative to the control groups. Inaddition, a markedly lower level of plasma insulin was observed duringOGTT in the ASO treatment group versus controls (5 ng/ml JNK ASO treatedvs 23 ng/ml saline treated).

An insulin tolerance test was also completed. There was an increase inrate and magnitude of glucose lowering after injecting insulin in theanimals treated with JNK antisense oligonucleotide. AUC is reduced byabout 50% by administration of JNK antisense oligonucleotide compared tosaline treated control. These data demonstrate that reduction of JNK1expression with JNK1 ASO significantly improved insulin sensitivity.

These date indicate that inhibition of JNK by antisense improves glucosetolerance and insulin sensitivity and, therefore, JNK1 antisenseoligonucleotides are useful for treating, preventing and/or amelioratingdisorders of or associated with glucose intolerance and/or insulinresistance, such as, for example, obesity, metabolic syndrome, diabetes,and hyperglycemia.

Liver Steatosis

To examine if the ASO treatment improved liver steatosis, both liver TGcontent and histology were analyzed.

Liver TG content was found to be greater than 40% lower in JNKASO-treated group than in controls in ob/ob mice (120 mg/g vs 200 mg/g).Histological examinations with both H&E staining and oil-red O stainingconfirmed a significant improvement in liver steatosis in JNK1 ASOtreated mice (much smaller and fewer fat droplets than those incontrols). In addition, the histological examination did not reveal anysign of ASO-related liver damage. Rather, improved liver steatosis wasaccompanied by improved liver function, as assessed by plasma ALT andAST measurements (133.2±10.1 U/L ALT vs 311.5±21.1 and 113.2±8.2 U/L ASTvs 187.8±14.6).

In addition to Liver steatosis, plasma transaminase levels and plasmacholesterol levels were improved for the treatment groups over thecontrol group.

As compared to controls, treatment with JNK1 ASO for 6 weeks loweredplasma total cholesterol levels by 40% in ob/ob mice in the fed state.Lipoprotein profile analysis confirmed that JNK1 ASO treatment loweredthe cholesterol content in all three major lipoprotein fractions, namelyVLDL-, LDL- and HDL-cholesterol.

Plasma triglycerides, total cholesterol, HDL-cholesterol,LDL-cholesterol, free fatty acids and transaminases are measured byroutine clinical analyzer instruments (e.g. Olympus Clinical Analyzer,Melville, N.Y.). Tissue triglyceride levels are measured using aTriglyceride GPO Assay from Roche Diagnostics (Indianapolis, Ind.).Liver triglyceride levels are used to assess hepatic steatosis, orclearing of lipids from the liver.

Hepatic steatosis is also assessed by routine histological analysis offrozen liver tissue sections stained with oil red O stain, which iscommonly used to visualize lipid deposits, and counterstained withhematoxylin and eosin, to visualize nuclei and cytoplasm, respectively.For H&E staining, liver, epididymal WAT and intrascapular BAT samplesfrom ob/ob mice were fixed in 10% buffered formalin and embedded inparaffin wax. For oil-red O staining, liver samples were collected inembedding medium. Multiple adjacent 4-μm sections were cut and mountedon glass slides. After dehydration, the sections were stained. Images ofthe histological sections were analyzed.

Metabolic Gene Expression

The ob/ob mice that received treatment were further evaluated at the endof the treatment period for the effects of target inhibition on theexpression genes that participate in lipid metabolism, cholesterolbiosynthesis, fatty acid oxidation, fatty acid storage, gluconeogenesisand glucose metabolism. Briefly, mRNA levels in liver and white andbrown adipose tissue were quantitated by real-time PCR as described inother examples herein, employing primer-probe sets that are generatedusing published sequences of each gene of interest. The observationsshowed 1) increased mRNA levels of adrenoceptor .beta.3 by >2-fold andUCP1 mRNA by >1.2-fold in BAT; 2) reduced mRNA levels of ACC1, ACC2,FAS, SCD1, DGAT-1 and DGAT-2 by 30-60% in WAT; and 3) reduced mRNAlevels of ACC1, FAS and G6Pase by >55%, and increased mRNA levels ofboth UCP2 and PPAR.alpha. by >2-fold in liver (see Table 35).

These data indicate that specific reduction of JNK1 expression with ASOsresults in increased fuel combustion and decreased lipogenesis in thismodel. Thus, JNK1 appears to play an important role in whole bodymetabolism and therapeutic inhibition of JNK1 in major metabolic tissuescould provide clinical benefit for obesity and metabolic syndrome.

Example 13 In vivo Studies in a Diet-Induced Model of Obesity (DIO)

To further confirm the metabolic effects of antisense suppression ofJNK1 expression, DIO mice were also treated with JNK1 antisenseoligonucleotides.

The C57BL/6 mouse strain is reported to be susceptible tohyperlipidemia-induced atherosclerotic plaque formation. Accordingly,these mice were fed a high-fat diet and used in the following studies toevaluate the effects of JNK1 antisense oligonucleotides on mRNAexpression in a model of diet-induced obesity.

Male C57BL/6J mice at 6 weeks of age were fed a diet containing 58 kcal% fat (Research Diet D12330, Research Diets Inc., New Brunswick, N.J.)for 15 weeks to induce obesity and insulin resistance. The animals werethen divided into different groups (n=6) and treated with JNK1 ASO orcontrol ASO at a dose of 25 mg/kg BW or with saline twice a week for 7weeks. Two JNK1 treatment oligonucleotides targeting two differentregion of JNK1 mRNA were used. The treatment oligonucleotides share thenucleobase sequence of SEQ ID NO.: 114, Isis No. 104492, orTGTTGTCACGTTTGCTTCTG, SEQ ID NO: 108, ISIS NO. 256463. Each were givenat the same dose. The control oligonucleotide is the same as describedabove. The treatment oligonucleotides are chimeric oligonucleotide 20nucleotides in length, targeted to a nucleic acid that encodes JNK1polypeptide. The nucleic acid encoding JNK1 polypeptide has a nucleosidesequence that is substantially similar to SEQ ID NO 87, 89, 90, 91.

During the treatment, weekly food intake and BW were monitored, and bodycomposition and other metabolic measurements were conducted (see below).At the end of the studies, animals were sacrificed. Blood samples werecollected by cardiac puncture, and tissues were dissected, weighed, andthen saved for further analysis.

mRNA Levels

Total RNA was isolated by homogenizing tissues in RLT buffer (Qiagen,Md.) followed by centrifugation with cesium chloride gradient. Real-timequantitative RT-PCR analysis was then performed to analyze the geneexpression. In DIO mice, treatment with 104492 reduced JNK1 mRNA by 78%,66% and 70% in liver, WAT and BAT, respectively. Treatment with ISIS256463 caused similar reduction of JNK1 expression in these tissues.

Plasma Glucose and Insulin Levels

Plasma insulin was measured with an insulin ELISA kit (ALPCODiagnostics, Windham, N.H.). Plasma glucose were measured with abiochemistry analyzer (Olympus AU400, Olympus American Inc, Melville,N.Y.).

Treatment with JNK1 ASO lowered plasma glucose and insulin levels inboth fed and fasted states when compared to controls (See Table 30).Treatment resulted in complete normalization of glucose levels andinsulin levels in DIO mice confirming the improved insulin sensitivityshown in the ob/ob model.

TABLE 30 Plasma Glucose and Insulin Levels in DIO Mice Saline ControlISIS 104492 Glucose Baseline 207.2 ± 8.2  200.8 ± 9.7  206.3 ± 7.8 (mg/dl) Fed 193.5 ± 9.8  189.0 ± 6.5  174.0 ± 4.2*  Fasting 141.0 ± 8.6 124.3 ± 13.0   76.5 ± 6.4** Insulin Baseline 2.23 ± 0.13 2.41 ± 0.342.26 ± 0.12 (ng/ml) Fed 2.51 ± 0.12 2.17 ± 0.39  0.75 ± 0.11** Fasting1.30 ± 0.50 0.92 ± 0.21  0.48 ± 0.05** Data are expressed as the mean ±SEM (n = 5-6). *P < 0.05 and **P < 0.01 when compared to either controlgroup.

Improved Hepatic Steatosis

Plasma transaminase (AST and ALT) activities were measured with abiochemistry analyzer (Olympus AU400, Olympus American Inc, Melville,N.Y.). Liver triglycerides (TG) was measured as previously described(Desai et al., 2001).

JNK1 ASO treatment lowered liver TG content by greater than 40% in DIOmice without causing liver toxicity, as assessed by plasma ALT and ASLactivities.

Plasma Cholesterol Levels

Total cholesterol and FFA concentrations were measured with abiochemistry analyzer (Olympus AU400, Olympus American Inc, Melville,N.Y.).

As compared to controls, treatment with JNK1 ASO for 6 weeks loweredplasma total cholesterol levels by about 35% in DIO mice in the fastedstate.

To investigate whether lowered plasma cholesterol levels with ASOtreatment in the ob/ob and DIO models were caused by reducing hepaticsynthesis and secretion, mouse primary hepatocytes were treated withJNK1 ASO and then de novo sterol synthesis was determined by measuringthe incorporation of [14C]-acetate into sterols. JNK1 ASO transfectedhepatocytes showed reduced de novo sterol synthesis by 13% as comparedto controls. Furthermore, gene expression analysis found that JNK1ASO-treated mice had significantly lower hepatic ApoB100 mRNA levelsversus controls. Reduction of ApoB100 expression has been welldemonstrated to reduce plasma cholesterol levels in rodents and severalother species. Therefore, without being bound by any theory, decreasedplasma cholesterol levels can be at least in part due to decreasedhepatic cholesterol output.

Feed Efficiency, Body Weight and Fat

In DIO mice, treatment with either of the two JNK1 oligonucleotides didnot result in a change in food intake compared to controls. Treatmentwith JNK1 ASOs lowered BW by greater than 10%, which resulted insignificant difference from the controls. Both JNK1 ASO-treated groupsalso showed greater than 35% lower epididymal fat depot weight andgreater than 20% lower percentage body fat content with no difference onlean body mass.

Metabolic rate was measured for a 24-h period using indirect calorimetry(Oxymax System, Columbus Instruments, Columbus, Ohio). JNK1 ASO-treatedmice had higher VO₂ than controls (about 12% or greater in the dark andabout 4% or greater in the light).

Expression of Metabolic Genes

The expression of representative metabolic genes in DIO mice wasanalyzed. Similar changes as seen in ob/ob mice were founding the DIOmouse model. Additionally, about a 70% increase in the expression ofboth UCP2 and UCP3 in WAT was found in JNK1 treated DIO mice versuscontrols (see Table 31), further indicating that reduction of JNK1expression not only inhibits lipogenesis but also increases metabolicrate.

Decreased De Novo Fatty Acid and Sterol Synthesis and Increased FattyAcid Oxidation

De novo fatty acid and sterol synthesis in transfected mouse hepatocyteswere determined by measuring the incorporation of [¹⁴C]acetate intofatty acids and sterols, respectively, as previously described (Jiang etal., 2005; Yu et al., 2005). Fatty acid oxidation was determined bymeasuring the oxidation of [¹⁴C]oleate into acid soluble products andCO₂ as described (Choi et al., 2007; Savage et al., 2006; Yu et al.,1997; Yu et al., 2005).

To confirm that the JNK1 ASO-caused changes in gene expressiontranslated into functional effects, cultured mouse primary hepatocyteswere transfected with JNK1 ASO and fatty acid oxidation and de novofatty acid synthesis were determined. Consistent with the changes ingene expression seen in vivo, fatty acid oxidation rate was about 35%higher or more whereas de novo fatty acid synthesis was about 20% lowerin JNK1 ASO transfected cells than controls.

Improved Insulin Signaling

In support of the increased insulin sensitivity seen in the insulin andglucose tolerance tests in Example 12 above and the fed/fasted glucoseand insulin measurements in both Example 12 and 13, mechanistic insulinsignaling assays were performed. The enhanced insulin sensitivityresulting from reduction of JNK1 expression was verified by analyzingthe activities of some key insulin signaling enzymes in both WAT andliver from DIO mice (treated with JNK1 ASO or control ASO and challengedwith insulin).

DIO mice were treated with JNK1 ASO or control ASO at a dose of 37.5mg/kg BW twice a week for 3 weeks. The mice were then fasted overnightand given a bolus i.p. injection of insulin at 2 U/kg BW or vehicle. Theanimals were then sacrificed, and liver and epididymal WAT werecollected and quickly frozen in liquid N₂ for further analysis. Equalamount of total proteins contained in pre-cleared fat or liverhomogenates were separated on gradient SDS-PAGE gels (BioRad, Hercules,Calif.) under reduced conditions and then transferred onto PVDFmembranes. The blots were then incubated with primary antibody againstAkt, Serine473-phosphorylated Akt (pAkt^(Ser437)) (Cell Signaling,Danvers, Mass.), or Ser302/307-phosphorylated IRS1 (pIRS1^(Ser302/307))(Biosource, Camarillo, Calif.). Signals were then detected by usingHRP-conjugated secondary antibody and ECL detection reagents (AmershanBiosciences).

A decreased level of pIRS^(Ser302/307) was found not only under basalconditions (without insulin challenge) but also after insulin challengein both tissues from JNK1 ASO-treated mice versus those from controlASO-treated mice. To evaluate whether decreased pIRS^(Ser302/307) causedincreased downstream insulin signaling activity, the level ofpAkt^(Ser473) in WAT was analyzed. A much higher level of pAkt^(Ser473)was found in JNK1 ASO-treated mice versus controls after insulinchallenge although its basal level was lower in the JNK1 ASO-treatedmice; the latter was probably due to the lower plasma insulin levels inthese mice. These data indicate that reduction of JNK1 expression withASO improved insulin signaling activity which supports at least in partthe increased insulin sensitivity detected in the tolerance tests andfed/fasted glucose and insulin measurements.

Statistical Analysis

Values presented represent the mean±SEM of three in vitro or 5-6 in vivoindependent measures per treatment. Statistical difference betweentreatment groups was determined using one-way ANOVA with Tukey HSDmultiple comparisons or two-tailed student t-test. P<0.05 was consideredto be significant.

TABLE 31 Metabolic Gene Expression in ob/ob and DIO Mice Liver WAT Genesaline control ASO JNK1 ASO saline control ASO JNK1 ASO ACL  100.0 ±11.0 90.2 ± 7.7 54.0 ± 6.6** ACC1  100.0 ± 11.0 81.2 ± 12  34.3 ± 1.2**100.0 ± 4.4  101 ± 9.8 39.2 ± 2.1** ACC2 100.0 ± 11  100.7 ± 15  121.1 ±15    100.0 ± 9.6 77.6 ± 7.9 38.1 ± 3.7** FAS  100.0 ± 13.2  79.4 ± 13.344.3 ± 3.2** 100.0 ± 4.1 110.7 ± 9.3  44.5 ± 3.7** Gyk 100.0 ± 6.2 106.3± 5.4  107.2 ± 6.4   100.0 ± 2.7 102.5 ± 4.6  65.8 ± 2.2** SCD1  100.0 ±28.4  78.3 ± 17.4 134.6 ± 12.3  100.0 ± 5.9 80.8 ± 4.3 45.3 ± 2.6**DGAT1 100.0 ± 4.0 93.4 ± 2.3 113.4 ± 6.4   100.0 ± 3.1 90.3 ± 4.1 67.2 ±5.1** DGAT2 100.0 ± 9.8 119.6 ± 6.2  128 ± 6.7  100.0 ± 4.1 87.6 ± 3.767.4 ± 2.7** HSL 100.0 ± 6.8 89.2 ± 6.2 91.1 ± 3.3  ATGL 100.0 ± 4.288.1 ± 4.8 79.9 ± 11.7  PPARα  100.0 ± 51.9 130.2 ± 28.9 212.8 ± 7.1** UCP2  100.0 ± 11.1 110.2 ± 21.2 209.8 ± 49.6** 100.0 ± 5.6  93 ± 5.1110.3 ± 6.9   ARβ₃ 100.0 ± 7.3 152.3 ± 20  143.8 ± 20.5  GK 100.0 ± 7.297.3 ± 5.4 146.1 ± 16.6*  G6Pase 100.0 ± 3.0 92.1 ± 6.7 44.6 ± 3.3** GS100.0 ± 4.5 107.4 ± 7.7  185.3 ± 16.6** PKCε 100.0 ± 8.9 84.2 ± 5.3 62.1± 8.3*  RBP4  100.0 ± 13.9 118.7 ± 7.3  63.2 ± 11.2* ApoB100  100.0 ±10.2 99.1 ± 8.9 75.1 ± 2.5*  The analysis was performed withquantitative RT-PCR. Total RNA was isolated from tissues of ob/ob micetreated with JNK1 ASO or control ASO at 25 mg/kg BW or with saline twicea week for 6 weeks. Data are expressed as the mean ± SEM (n = 5-6). *P <0.05 and **P < 0.01 when compared to either control group.

1-10. (canceled)
 11. A method of treating diabetes in a subjectcomprising administering to said subject a pharmaceutical compositioncomprising a glucose-lowering agent and a therapeutically effectiveamount of an antisense compound targeted to a JNK1 nucleic acid.
 12. Amethod of treating diabetes in a subject comprising administering tosaid subject a glucose-lowering agent and a therapeutically effectiveamount of an antisense compound targeted to a JNK1 nucleic acid.
 13. Themethod of claim 12, wherein the administering thereby reduces glucoselevels or improves insulin sensitivity or both.
 14. The method of claim12, wherein said glucose-lowering agent is insulin or an insulin-analog,a biguanide, a meglitinide, a thiazolidinedione, sulfonylurea or analpha-glucosidase inhibitor.
 15. The method of claim 14, wherein theglucose lowering agent is a biguanide.
 16. The method of claim 15,wherein the biguanide is metformin.
 17. The method of claim 14, whereinthe glucose lowering agent is a meglitinide.
 18. The method of claim 17,wherein the meglitinide is nateglinide or repaglinide.
 19. The method ofclaim 14, wherein the glucose lowering agent is a thiazolidinedione. 20.The method of claim 19, wherein the thiazolidinedione is nateglinidepioglitazone, rosiglitazone, or troglitazone.
 21. The method of claim20, wherein the glucose lowering agent is a rosiglitazone.
 22. Themethod of claim 21, wherein blood glucose levels are decreased and bodyweight is maintained or reduced.
 23. The method of claim 14, wherein theglucose lowering agent is an alpha-glucosidase inhibitor.
 24. The methodof claim 23, wherein the alpha-glucosidase inhibitor is acarbose ormiglitol.
 25. The method of claim 14, wherein the glucose lowering agentis sulfonylurea. 26-37. (canceled)
 38. The method of claim 12 whereinthe glucose lowering agent and antisense compound are administeredconcomitantly.
 39. The method of claim 12, wherein the administeringcomprises parenteral administration.
 40. The method of claim 39, whereinthe parenteral administration comprises subcutaneous or intravenousadministration.
 41. (canceled)
 42. (canceled)
 43. The method of claim12, wherein the antisense compound has at least 95% complementarity toSEQ ID NO: 87, 89, 90 or
 91. 44. The method of claim 12, wherein theantisense compound has 100% complementarity to SEQ ID NO: 87, 89, 90 or91.
 45. The method of claim 12, wherein the antisense compound is 12 to30 nucleosides in length. 46-48. (canceled)
 49. The method of claim 12,wherein the antisense compound is an antisense oligonucleotide.
 50. Themethod of claims 12, wherein the antisense compound comprises at leastone modified sugar moiety.
 51. The method of claim 50, wherein themodified sugar moiety is a 2′-O-methoxyethyl sugar moiety. 52-58.(canceled)